An Introduction To Agricultural Biochemistry

AN INTRODUCTION
TO AGRICULTURAL
BIOCHEMISTRY
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AN INTRODUCTION
TO AGRICULTURAL
BIOCHEMISTRY
J.M. Chesworth
Honorary Lecturer in Agricultural Biochemistry and Nutrition
Department of Agriculture
University of Aberdeen, UK
and
Independent Consultant
Mar Technical Services
Huntly, UK
T. Stuchbury
Lecturer in Agricultural Biochemistry and Plant Physiology
and
J.R. Scaife
Lecturer in Agricultural Biochemistry and Nutrition
CHAPMAN & HALL

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First edition 1998
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CONTENTS
Preface XVlll
List of abbreviations xix
PART ONE: THE CELL AND CELLULAR CONSTITUENTS 1
1 Cell structure and function 3

1.1 Introduction 3
1.2 Components of cells 3
1.2.1 Plasma membrane 3
1.2.2 Cytoplasm 4
1.2.3 The nucleoid and nucleus 6
1.2.4 Cell walls 6
1.2.5 Ribosomes 6
1.2.6 Endoplasmic reticulum 6
1.2.7 Vacuoles and specialized vesicles 7
1.2.8 Mitochondria 8
1.2.9 Chloroplasts 8
1.2.10 Cytoskeleton 8
1.3 Cell specialization and interaction 9
2 Water and solutions 11

2.1 Introduction 11
2.2 The ionization of water 12
2.2.1 The pH of water 12
2.3 What are acids and bases? 14
2.4 Biological systems, ionic strength and pH 14
2.4.1 Stabilization of pH by buffers 14
2.5 Colligative properties 15
2.5.1 Depression of freezing point 16
2.5.2 Osmotic pressure 16
2.5.3 Semipermeable membranes that allow some solutes to pass 17
3 The carbohydrates 19
3.1 Introduction 19
3.2 Structures of sugars 20
3.2.1 Optical isomers 20
3.3 Naming of sugars 20
3.4 Sugars with four carbon atoms, the tetroses 20
vi Contents
3.5 Sugars with five carbon atoms, the pentoses 21
3.5.1 Ring formation in sugars 22
3.5.2 Five- and six-membered rings 23
3.5.3 Ring formation is not permanent 23
3.6 Sugars with six carbon atoms, the hexoses 23
3.6.1 Glucose 23
3.6.2 Fructose 23
3.6.3 Other hexoses 23
3.7 Reducing and non-reducing sugars 24
3.8 Formation of sugar acetals 24
3.8.1 Formation of disaccharides 25
3.8.2 Sucrose 25
3.9 Polysaccharides 26
3.9.1 The storage carbohydrates - starch and glycogen 26
3.9.2 Structural polysaccharides in plants 29
3.9.3 Other polysaccharides and related compounds 32
4 Fatty acids and lipids 35
4.1 Introduction 35
4.2 Structure and occurrence of lipids 35
4.2.1 Fatty acids 35
4.2.2 Triacylglycerols and other acylglycerols 39
4.2.3 Glycerophospholipids 42
4.2.4 Glycosylglycerides 43
4.2.5 Sphingolipids 44
4.2.6 Terpenes and steroids 45
4.2.7 Waxes 47
5 Amino acids and proteins 51
5.1 Introduction 51
5.2 Amino acids 52
5.2.1 Structure of amino acids 52
5.3 Non-protein amino acids and related compounds 52
5.3.1 Canavanine 52
5.3.2 Selenium-containing amino acids 52
5.3.3 Mimosine 54
5.3.4 Lathyrogens 55
5.3.5 S-methyl cysteine sulphoxide (SMCO) 55
5.3.6 Alkaloids 55
5.4 Phenolics 57
5.4.1 Lignin 57
5.4.2 Tannins 57
5.4.3 Flavonoids 58
5.5 Peptide bonds 59
5.6 Protein function and structure 61
5.6.1 Primary protein structure 62
5.6.2 Secondary protein structure 62
5.6.3 Tertiary structure 63
Contents vii
5.6.4 Quaternary structure 64
5.7 Properties of proteins 66
5.7.1 Ionic strength and presence of specific ions 67
5.7.2 Effect of pH 68
5.7.3 Denaturation 68
5.7.4 Effect of heat 69
5.8 Prions 69
6 Enzymes 71
6.1 Introduction 71
6.2 Types of reactions catalysed by enzymes 71
6.3 Mode of action of enzymes 72
6.4 Factors contributing to enzyme activity 73
6.4.1 Proximity of substrates at the active site 73
6.4.2 Environment 74
6.4.3 Acid-base catalysis 74
6.4.4 Effects on the stability of substrates and reaction intermediates 74
6.4.5 Formation of covalent enzyme-substrate intermediates 74
6.5 Factors affecting the rates of enzyme-catalysed reactions 76
6.5.1 Enzyme concentration 76
6.5.2 Substrate concentration 76
6.5.3 Temperature 79
6.5.4 pH 79
6.5.5 Presence of inhibitors 81
6.5.6 Presence of coenzymes 84
6.6 Allosteric enzymes 90
6.7 Molecular recognition 91
6.7.1 Receptors 91
6.7.2 Antibodies 91
7 Purines, pyrimidines and nucleic acids 95
7.1 Introduction 95
7.2 Purines and pyrimidines 95
7.3 Deoxyribonucleic acid (DNA) 96
7.3.1 Chemical nature of DNA 96
7.3.2 The DNA double helix 98
7.3.3 Structure of DNA in prokaryotes and eukaryotes 98
7.3.4 Organelle DNA 100
7.4 Ribonucleic acid (RNA) 100
7.4.1 Messenger RNA (mRNA) 101
7.4.2 Transfer RNA (tRNA) 101
7.4.3 Ribosomal RNA (rRNA) 102
8 Vitamins and minerals 105
8.1 Vitamins in biochemistry 105
8.1.1 Introduction 105
8.1.2 Thiamin (vitamin B1) 106
8.1.3 Riboflavin (vitamin B2) 108
viii Contents
8.1.4 Nicotinic acid (niacin, formerly called vitamin B5) 108
8.1.5 Pantothenic acid 112
8.1.6 Pyridoxine, pyridoxal and pyridoxamine (vitamin B6) 112
8.1.7 Biotin 114
8.1.8 Folic acid 114
8.1.9 Vitamin B12 115
8.1.10 Vitamin C 117
8.1.11 Choline 118
8.1.12 Carnitine 119
8.1.13 Vitamin A 119
8.1.14 Vitamin 0 121
8.1.15 Vitamin E 124
8.1.16 Vitamin K 126
8.2 Minerals in biochemistry 127
8.2.2 Calcium 127
8.2.3 Phosphorus 128
8.2.4 Magnesium 128
8.2.5 Sodium, chloride and potassium 129
8.2.6 Sulphur and iron 129
8.2.7 Other elements with known biochemical functions 130
9 The composition of agricultural products 133
9.1 Introduction 133
9.1.1 Energy storage in animals and plants 133
92 The composition of animals 134
9.2.1 Body composition 134
9.2.2 Milk 134
9.3 Plant materials 134
9.4 Principal nutrients in plants and animals 00
9.4.1 Proteins 00
9.4.2 Lipids 00
9.4.3 Carbohydrates 00
PART TWO: METABOLISM 139

10 Glycolysis 141
10.2 Stage 1 - preparing glucose for splittil)g into two three-carbon units 141
10.2.1 Glucose phosphorylation 141
10.2.2 Fructose and its phosphates 143
10.2.3 Splitting of fructose-1,6-bisphosphate 143
10.3 Stage 2 - metabolism of the three-carbon compounds 143
10.3.1 First oxidation step 143
10.3.2 First energy released in the form of ATP 145
10.3.3 Formation of pyruvate 145
10.4 The entry of other sugars 145
10.4.1 Fructose 146
Contents ix
10.4.2 Galactose 146
10.4.3 The entry of glycogen 146
11 The tricarboxylic acid cycle 149
11.2 The reactions of the TCA cycle 149
11.2.1 Production of acetyl-CoA 149
11.2.2 Reactions leading to the production of CO 2 150
11.2.3 Reactions leading back to the formation of oxaloacetate 150
11.2.4 Overall reactions of the tricarboxylic acid cycle 153
11.3 Links with other metabolic pathways 153
11.4 Replenishment of TCA cycle intermediates 155
11.5 Conversion of propionate to glucose via the TCA cycle 155
11.6 Regulation of the TCA cycle 155
11.7 The glyoxylate cycle 156
12 Electron transport and oxidative phosphorylation 161
12.2 The mitochondrion 163
12.3 Components of the electron transport chain 163
12.3.1 Flavoproteins 163
12.3.2 The iron-sulphur proteins 164
12.3.3 Ubiquinone 164
12.4 The electron transport chain complexes 164
12.4.1 Complex I - the NADH-dehydrogenase complex 164
12.4.2 Complex II - the succinate dehydrogenase complex 164
12.4.3 Complex III - the cytochrome b, c1 complex 165
12.4.4 Complex IV - cytochrome oxidase 165
12.5 Coupling of electron transport to ATP synthesis 166
12.6 The yield of ATP 168
12.7 NADH produced in the cytoplasm enters the electron transport chain via 169
shuttle reactions
12.8 Regulation of oxidative phosphorylation by ADP/ATP supply 169
13 Fatty acid oxidation and lipid breakdown 173
13.2 j3-0xidation 174
13.2.1 Mitochondrialj3-oxidation in animal tissues 174
13.2.2 The reactions of j3-oxidation 176
13.2.3 j3-0xidation of odd-numbered acids 176
13.2.4 j3-0xidation of unsaturated acids 178
13.2.5 j3-0xidation in peroxisomes and glyoxisomes 179
13.2.6 The formation of ketone bodies 181
13.3 a-Oxidation 182
13.4 w-Oxidation 183
13.5 Peroxidation of fatty acids 183
13.5.1 Chemistry of lipid peroxidation 184
13.5.2 Prevention of fatty acid peroxidation 185
x Contents
13.5.3 Detection and measurement of lipid peroxidation 185
13.5.4 Effects of peroxidation in living organisms 186
13.5.5 Lipoxygenase and cyclo-oxygenase 188
13.6 Breakdown of lipids 189
13.6.1 Triacylglycerol breakdown 190
13.6.2 Phospholipid breakdown 191
13.6.3 Breakdown of glycolipids 192
14 Breakdown of proteins and the oxidation of amino acids 193
14.2 Breakdown of proteins 194
14.2.1 Digestion of proteins 194
14.2.2 Protein turnover 194
14.3 Breakdown of amino acids 194
14.3.1 Transamination reactions 194
14.3.2 Deamination 195
14.3.3 Oxidation of carbon skeletons of amino acids 195
14.3.4 The fate of ammonia 197
14.3.5 The urea cycle 197
14.3.6 The fate of urea in ruminants 199
14.4 Precursor functions of amino acids 199
15 The pentose phosphate pathway 201
15.2 Oxidative reactions 201
15.3 Rearrangement reactions 201
15.4 Importance of the pathway 205
15.5 Regulation of the pathway 206
16 Fermentation pathways 207
16.2 Anaerobic environments in agriculture 207
16.3 Lactate production 207
16.3.1 Muscle metabolism 208
16.3.2 Regeneration of glucose 208
16.4 Animal digestive systems 208
16.4.1 Acetate formation 209
16.4.2 Propionate formation 209
16.4.3 Butyrate synthesis 210
16.5 Soils 210
16.6 Waste treatment 211
16.7 Methane production 213
16.8 Dairy products 213
16.9 Meat 213
16.10 Fermentation in herbages 214
16.11 Ethanol production 214
17 Photosynthesis 217
Contents xi
17.2 Chloroplasts 217
17.3 The light reactions 217
17.3.1 Photosynthetic pigments 217
17.3.2 Light absorption 219
17.3.3 Resonance energy transfer 221
17.3.4 The electron transport system 221
17.3.5 Photosystem I 222
17.3.6 Photosystem II 223
17.3.7 Cytochrome b-f complex 225
17.3.8 Plastocyanin 225
17.4 Integration of the electron transport system 225
17.5 ATP production 225
17.6 The dark reactions (Calvin cycle) 226
17.7 Control of photosynthesis 229
17.8 Photorespiration 229
17.8.1 Factors affecting rates of photorespiration 230
17.9 Photosynthesis in C4 plants 232
17.10 Crassulacean acid metabolism 233
17.11 Herbicides and photosynthesis 234
18 Gluconeogenesis and carbohydrate synthesis 237
18.1.1 Starting materials 237
18.1.2 Outline of the pathway 238
18.1.3 Differences between gluconeogenesis and glycolysis 238
18.2 Gluconeogenesis via pyruvate 238
18.2.1 The control of pyruvate production and use 238
18.3 The production of fructose-1,6-bisphosphate 238
18.4 The hydrolysis of fructose-1,6-bisphosphate 240
18.4.1 The control of phosphofructokinase and fructose-1,6-bisphosphatase 240
18.4.2 The fate of fructose-6-phosphate 242
18.5 The utilization of glucose-6-phosphate 243
18.6 Gluconeogenesis from propionate 243
18.7 The synthesis of complex carbohydrates 243
18.7.1 Disaccharides 243
18.7.2 Synthesis of polysaccharides 246
19 Synthesis of fatty acids and lipids 251
19.2 Tissue and subcellular location of fatty acid synthesis 252
19.3 Source of the primary substrate - acetate 252
19.4 Production of malonyl-CoA 253
19.4.1 Animals 254
19.4.2 Plants 256
19.4.3 Bacteria 256
19.5 Synthesis of long-chain saturated fatty acids from acetyl-CoA and malonyl-CoA 256
19.5.1 Acyl carrier protein and its function 256
19.5.2 The reactions of fatty acid synthesis 257
Xll Contents
19.5.3 Chain length specificity of fatty acid synthetases 259
19.5.4 Synthesis of branched-chain fatty acids 260
19.5.5 Release of fatty acids from fatty acid synthetase 261
19.6 Fatty acid elongation 261
19.7 Formation of unsaturated fatty acids 262
19.7.1 Desaturation of fatty acids in animals 262
19.7.2 Desaturation of fatty acids in plants 263
19.7.3 Essential fatty acids 263
19.8 Synthesis of triacylglycerols 265
19.8.1 The 2-monoacylglycerol pathway 265
19.8.2 The glycerol-3-phosphate pathway 266
19.8.3 Triacylglycerol synthesis in plants 267
19.9 Phospholipids 268
19.10 Glycolipid synthesis 268
19.11 Synthesis of sphingolipids 270
19.12 Biosynthesis of terpenes and sterols 271
19.12.1 Synthesis of mevalonic acid 271
19.12.2 Conversion of mevalonic acid to squalene 273
20 Synthesis of amino acids 279
20.2 Assimilation of nitrate 279
20.3 Nitrogen fixation 281
20.3.1 Molecular biology of nitrogen fixation 282
20.4 Assimilation of ammonia 282
20.5 Biosynthesis of amino acids 284
20.5.1 Aromatic amino acids and related compounds 284
20.5.2 Branched-chain aliphatic amino acids 285
20.6 Nutritional role of amino acids 287
20.7 Herbicides and amino acid biosynthesis 289
21 The synthesis of nucleic acids and proteins 291
21.2 Synthesis of purine and pyrimidine nucleotides 292
21.3 Replication of DNA 292
21.3.1 DNA synthesis in viruses 294
21.3.2 Accuracy of DNA synthesis 296
21.4 Synthesis of RNA 296
21.5 The genetic code 297
21.6 Protein synthesis 297
21.6.1 Amino acid activation 297
21.6.2 Initiation 299
21.6.3 Elongation 300
21.6.4 Termination 300
21.6.5 Post-translational modification of proteins 300
21.6.6 Location of protein synthesis 301
21.7 Regulation of protein synthesis 303
21.7.1 Regulation in prokaryotes 303
Contents xiii
21.7.2 Regulation in eukaryotes 305
21.8 Protein synthesis in chloroplasts and mitochondria 306
21.9 Genetic engineering 308
21.9.1 Enzymes used in DNA manipulation 308
21.9.2 Isolation and synthesis of DNA 308
21.9.3 Gene cloning 309
21.9.4 Screening techniques 310
21.9.5 Restriction fragment length polymorphism (RFLP) 310
21.9.6 Antisense RNA 310
21.9.7 Vectors and methods for insertion of DNA into cells 310
21.9.8 Site-specific mutagenesis (oligonucleotide-directed mutagenesis) 311
22 Compartments, membranes and regulation 313
22.1 Cell compartments 313
22.2 Lipids and membranes 313
22.3 Transport across membranes 315
22.3.1 Membrane transport mechanisms 316
22.3.2 Other functions of membranes 321
22.4 Principles of metabolic regulation 322
22.4.1 Allosteric regulation 323
22.4.2 Covalent modification 323
22.4.3 Changes in the amount of enzyme 324
22.4.4 Coordinated regulation of pathways 325
22.4.5 Coordination of metabolism in different tissues 328
PART THREE: STRATEGIES FOR PROCESSING OF NUTRIENTS IN PLANTS 333

23 Seeds and germination 335
23.1 Seeds and plant development 335
23.2 Seeds as food and agricultural commodities 335
23.3 Seed composition 336
23.3.1 Seed carbohydrates 336
23.3.2 Seed lipids 337
23.3.3 Seed proteins 337
23.3.4 Seed minerals 339
23.4 Germination 340
23.4.1 Starch breakdown 340
23.4.2 Beer and whisky production 341
23.4.3 Protein breakdown 342
23.4.4 Lipid breakdown 3·!2
24 Vegetative growth of plants 343
24.2 Composition of shoots 344
24.2.1 Plant cell walls 344
24.3 Important shoot crops and their products 347
24.3.1 Temperate grasses 347
24.3.2 Tropical grasses 348
xiv Contents
24.3.3 Forage legumes 348
24.3.4 Brassicas 348
24.3.5 Straws 348
24.3.6 Hay 348
24.3.7 Silage 349
24.3.8 Wood 349
24.4 Important root and tuber crops 351
24.4.1 Root crops 351
24.4.2 Tubers 351
25 Reproductive growth 353
25.1 Flowering 353
25.2 Fruit development and composition 353
25.3 Fruit ripening 354
25.3.1 Changes in colour 354
25.3.2 Changes in texture 354
25.3.3 Changes in flavour 355
25.3.4 Respiration in ripening fruit 355
25.4 Seed development 356
25.4.1 Starch biosynthesis 356
25.4.2 Protein synthesis 357
25.4.3 Biosynthesis of fats 357
26 Plant nutrition 359
26.2 Biochemical functions of major plant nutrients 359
26.2.1 Nitrogen 359
26.2.2 Sulphur 359
26.2.4 Potassium 362
26.2.5 Calcium 362
26.2.6 Magnesium 362
26.3 Trace elements - micronutrients 362
26.4 Toxic effects of minerals 362
26.5 Interaction between carbon and nitrogen metabolism 363
26.5.1 Carbon assimilation 364
26.5.2 Nitrogen assimilation 364
26.5.3 Senescence and nutrient cycling 366
27 Regulation of plant growth and development 368
27.2 Responses to light 368
27.2.1 Effects on photosynthesis 368
27.2.2 Phytochrome-mediated responses 368
27.2.3 Other responses to light 370
27.3 Responses to temperature 370
27.3.1 Photosynthesis 370
27.3.2 Vernalization 370
Contents xv
27.4 Responses to atmosphere 370
27.5 Responses to stress 371
27.5.1 Temperature stress 371
27.5.2 Water stress 373
27.5.3 Salt stress 373
27.6 Nature of plant hormones 374
27.7 Auxins 374
27.7.1 Biochemistry of auxins 374
27.7.2 Synthetic auxins 375
27.7.3 Sites of synthesis and transport of auxins 376
27.7.4 Physiological activities and applications of auxins 377
27.8 Gibberellins 379
27.8.1 Biochemistry of gibberellins 379
27.8.2 Sites of synthesis and transport of gibberellins 379
27.8.3 Physiological activities and applications of gibberellins 379
27.8.4 Growth retardants 382
27.9 Cytokinins 384
27.9.1 Biochemistry of cytokinins 384
27.9.2 Sites of synthesis and transport of cytokinins 384
27.9.3 Physiological activities and applications of cytokinins 384
27.10 Abscisic acid (ABA) 387
27.10.1 Biochemistry of abscisic acid 387
27.10.2 Sites of synthesis and transport of abscisic acid 387
27.10.3 Physiological activities and applications of abscisic acid 387
27.11 Ethylene 388
27.11.1 Biochemistry of ethylene 388
27.11.2 Sites of synthesis and transport of ethylene 389
27.11.3 Methods of modulating the effects of ethylene on plants 389
27.11.4 Physiological activities and applications of ethylene 389
27.12 Miscellaneous plant growth regulators 392
27.12.1 Morphactins 392
27.12.2 Maleic hydrazide 392
27.12.3 Glyphosine 392
PART FOUR: STRATEGIES FOR PROCESSING OF NUTRIENTS IN ANIMALS 393

28 Digestion and absorption in ruminants and non-ruminants 395
28.2 Structure of the digestive tract 395
28.3 Carbohydrate digestion in monogastric animals 396
28.4 Carbohydrate digestion in ruminants 398
28.5 Absorption and utilization of glucose 398
28.6 Digestion of lipids in monogastric animals 399
28.6.1 Digestion in the stomach 399
28.6.2 Digestion in the small intestine 400
28.7 Absorption of lipid from the small intestine 402
28.7.1 Glycerides and fatty acids 402
XVi Contents
28.7.2 Phospholipids 403
28.7.3 Cholesterol 403
28.7.4 Chylomicron formation 403
28.8 Uptake of absorbed lipid by body tissue 403
28.9 Digestion of lipids in ruminant animals 404
28.10 Lipid digestion in poultry 406
28.11 Digestion of proteins in monogastric animals 406
28.12 Digestion of protein in ruminants 409
28.13 Inhibitors of digestive enzymes 410
29 Maintenance 413
29.2 Body temperature and heat production 413
29.3 Biochemical production of heat 414
29.3.1 Background heat production from the maintenance of ion gradients 415
in cells
29.3.2 Background heat production from protein turnover 416
29.3.3 Background heat production from other metabolic events 417
29.3.4 Heat production from muscular activity 418
29.3.5 Heat production from uncoupled phosphorylation 419
30 Regulation and manipulation of growth and development in animals 421
30.1.1 General principles 422
30.2 Rates and patterns of growth 422
30.3 Muscle growth 424
30.3.1 Cellular growth as a component of muscular growth 424
30.3.2 Protein accretion as a component of growth 425
30.3.3 Control of protein synthesis 425
30.4 Growth of collagen 425
30.5 Growth of bone 427
30.5.1 Calcification of bone 428
30.6 Growth of adipose tissue 429
30.6.1 Growth in adipose tissue cell number 429
30.6.2 Deposition of fat within adipocytes 430
30.7 Manipulation of growth 431
30.7.1 Growth hormone and insulin-like growth factors 431
30.7.2 Oestrogens and androgens 433
30.7.3 The j3-agonists 435
30.7.4 Glucocorticoids 439
30.7.5 Thyroid hormones 439
30.7.6 Antibiotics 440
31 Lactation and its manipulation 447
31.2 Origins of the components of milk. 448
Contents xvii
31.3 The origin of lactose 449
31.4 Milk proteins 451
31.4.1 The origin of milk proteins 451
31.4.2 Amino acid supply to the mammary gland 451
31.5 The fats 452
31.5.1 Synthesis de novo 452
31.5.2 Uptake of fatty acids from blood 453
31.5.3 Modifications to fatty acids in the mammary gland 453
31.6 The supply of energy in the mammary gland 454
31.7 Metabolism in lactation. 454
31.7.1 Endocrine control of lactation 454
31.8 Manipulation of lactation 455
31.8.1 Dietary manipulation of lactation 456
31.8.2 Manipulation of milk production by exogenous hormones 457
32 Muscle and meat 459
32.2 Biochemistry of muscular contraction 459
32.2.1 Structure of thick filaments 461
32.2.2 Structure of thin filaments 461
32.2.3 Mechanism of muscle movement 461
32.2.4 Control of muscle movement 462
32.3 Energy provision in muscle tissue 464
32.3.1 Myoglobin 465
32.4 Changes in muscle after death 465
32.4.1 Enzymes leading to the degeneration of myofibrils 466
32.4.2 The role of myoglobin 466
32.4.3 Conditioning 467
32.4.4 Cold shortening 467
32.4.5 Effects of stress pre-slaughter 467
PREFACE
Biochemistry books tend to fall into two distinct The book is divided into four parts. Part 1,
categories: specialist volumes written for the The Cell and Cellular Constituents, and Part 2,
researcher that deal in great depth with narrow Metabolism, provide a traditional treatment of
areas of the subject, and large, comprehensive the structure and function of biological mole-
textbooks that aim to give an overall coverage cules and how they are synthesized and
of the material. Many of the latter emphasize degraded, but have been written with an agri-
areas of particular interest to students of medi- cultural flavour, drawing on examples from
cine and related disciplines. By their emphases plants and animals of agricultural importance.
and their very size they sometimes prove Parts 3 and 4 are more of a departure from
daunting to students from other disciplines. most biochemistry books. Part 3 is devoted to
In writing this book our main aim was to plants. It looks at the important stages in plant
provide an introduction to biochemical princi- growth and development and examines the
ples for students of agriculture, but we hope biochemical process which underpin them; it
that it also proves a useful text for students of also discusses the regulation and opportunities
forestry, veterinary science and other areas of for manipulation of such processes. Part 4 con-
applied biology. These students, for whom centrates on animals and bridges the gap
biochemistry may not be the primary interest, between biochemistry and nutrition. It exam-
need to understand how organisms function ines the ways in which animals acquire nutri-
at a chemical level and how this relates to the ents through the digestive tract and looks at the
more complex biological systems which they biochemical basis of maintenance, growth, lac-
are studying. We have attempted to target tation and meat production. More specialized
those areas of biochemistry which are of most texts are available for the student who wants a
relevance and to keep coverage of the subject more in-depth treatment of these areas.
simple. The book is intended for use principal- Discussions with many of our colleagues
ly in first- and second-year courses where bio- have contributed to the completion of this book
chemistry is first introduced to students. and to them we extend our thanks. Greatest
Those students who find the subject interest- thanks must go to our wives who have shown
ing and wish to delve deeper are provided remarkable patience and understanding during
with references to more detailed texts. the preparation of the manuscript.
ABBREVIATIONS
2,4,5-T (2,4,5-trichlorophenoxy)acetic acid DNA deoxyribonucleic acid

2,4-0 (2,4-dichlorophenoxy)acetic acid DUP digestible undegradable protein
ABA abscisic acid EFA essential fatty acid
ACC I-aminocyclopropane I-carboxylic acid EPA eicosapentaenoic acid
ACP acyl carrier protein EPSP synthase 5-enolpyruvylshikimic acid-
ADP adenosine diphosphate 3-phosphate synthase
AIDS acquired immune deficiency syndrome ER endoplasmic reticulum
ALA o-aminolevulinic acid ES enzyme-substrate complex
a-LA a-lactalbumin F-2,6-P fructose-2,6-bisphosphate
AMP adenosine monophosphate F-6-P fructose-6-phosphate
AOA aminooxyacetic acid f-Met N-formyl methionine
APS adenosine-5' -phosphosulphate FAD flavin adenine dinucleotide (oxidized)
ATP adenosine triphosphate FADH2 flavin adenine dinucleotide
ATPase enzyme that hydrolyses ATP (reduced)
AVG aminoethoxyvinylglycine Fd/FdH2 ferredoxin (oxidized and reduced
BA benzyladenine forms)
BAT brown adipose tissue FIGLU formiminoglutamic acid
BCCP biotin carboxyl carrier protein FLKS fatty liver and kidney syndrome
I3-LG 13 lactoglobulin FMN flavin mononucleotide (oxidized)
BSE bovine spongiform encephalopathy FMNH2 flavin mononucleotide (reduced)
bST bovine somatotropin FPP farnesyl pyrophosphate
CAM crassulacean acid metabolism FW fresh weight
cAMP 3',5' -cyclic adenosine monophosphate GA gibberellic acid
CCC chlorocholine chloride (cycocel) GOP guanosine diphosphate
CCN cerebrocortical necrosis GH growth hormone
cDNA complementary deoxyribonucleic acid GMP guanosine-5' -monophosphate
COP cytidine diphosphate GOGAT glutamate synthase
CMP cytidine mono phosphate GTP guanosine triphosphate
CoA coenzyme A GS glutamine synthetase
CTP cytidine 5' -triphosphate HMG-CoA hydroxymethylglutaryl
Cyt cytochrome coenzyme A
DAG diacylglycerol hnRNA heterogeneous nuclear ribonucleic
DFD dark firm dry (of meat) acid
aG free energy change for reaction HPLC high performance liquid chromatog-
DGDG digalactosyldiacylglycerol raphy
DHA docosahexaenoic acid 1M indole-3-acetic acid
OM dry matter IBA indole butyric acid
DMAPP dimethylallyl pyrophosphate IGF-I insulin-like growth factor-I
xx Abbreviations
IGF-II insulin-like growth factor-2 PGF2 prostaglandin F2
IGFs insulin-like growth factors PGR plant growth regulator
IMP inosine monophosphate Pj orthophosphate
IPP isopentenyl pyrophosphate PI phosphatidylinositol
kDa kilodaltons PME pectin methylesterase
kJ kilojoule (joule x 103) PMF proton motive force
Km Michaelis constant PP j pyrophosphate
LHC light-harvesting complex Pq plastoquinone
Met methionine Pr/Pfr phytochrome (red and far-red absorb-
MGDG monogalactosyldiacylglycerol ing forms)
MJ megajoule (joule x 106) PRPP phosphoribosyl pyrophosphate
mRNA messenger ribonucleic acid PS phosphatidylserine
NAA a-naphthalene acetic acid PSE pale soft exudative (of pig meat)
NAD+ nicotine adenine dinucleotide (oxi- PSI photosystem 1
dized) PSII photo system 2
NADH nicotine adenine dinucleotide PSS porcine stress syndrome
(reduced) PUFA polyunsaturated fatty acid
NADP nicotine adenine dinucleotide phos- PV peroxide value
phate (oxidized) RET resonance energy transfer
NADPH nicotine adenine dinucleotide RFLP restriction-fragment length polymor-
phosphate (reduced) phism
NEFA non-esterified fatty acid RNA ribonucleic acid
NPN non-protein nitrogen rRNA ribosomal ribonucleic acid
OxAc oxaloacetate Rubisco ribulose-1,5-bisphosphate carboxy-
P680, P700 reaction centres of photosystem II lase/oxygenase
and photosystem I, respectively S Svedberg
PA phosphatidic acid SAM S-adenosylmethionine
PABA p-aminobenzoic acid SMCO S-methyl cysteine sulphoxide
PAL phenylalanine ammonia lyase snRNP small nuclear ribonuclear particle
PC phosphatidykholine SRP signal recognition particle
Pc plastocyanin TCA cycle tricarboxylic acid cycle
PCR polymerase chain reaction TPP thiamin pyrophosphate
PDH pyruvate dehydrogenase tRNA transfer ribonucleic acid
PE phosphatidylethanolamine UDP uridine diphosphate
PEM polioencephalomalacia UDPG uri dine diphosphate glucose
PEP phosphoenolpyruvate UMP uri dine monophosphate
PES prostaglandin endoperoxide synthetase UTP uridine triphosphate
PFK1 phosphofructokinase 1 UV ultraviolet
PFK2 phosphofructokinase 2 VFA volatile fatty acid
PG phosphatidylglycerol VLDL very low density lipoprotein
PGEz prostaglandin E2
PART ONE
THE CELL AND CELLULAR CONSTITUENTS

CELL STRUCTURE AND FUNCTION 1
1.1 Introduction 3
1.2 Components of cells 3
1.2.1 Plasma membrane 3
1.2.2 Cytoplasm 4
1.2.3 The nucleoid and nucleus 6
1.2.4 Cell walls 6
1.2.5 Ribosomes 6
1.2.6 Endoplasmic reticulum 6
1.2.7 Vacuoles and specialized vesicles 7
1.2.8 Mitochondria 8
1.2.9 Chloroplasts 8
1.2.10 Cytoskeleton 8
1.3 Cell specialization and interaction 9
1.1 INTRODUCTION nuclear envelope, are defined as eukaryotic.

The cell is the unit from which living organ- Brown, red and green algae, protozoa, fungi
isms are built. Despite the wide diversity of and multicellular plants and animals consist of
eukaryotic cells. They have developed an
organisms, the cells of which they are com-
posed have many common features and most internal system of membranes that separates
carry out very similar biochemical processes. the cell into distinct areas, called organelles,
The cell consists of a plasma membrane sur- which have specific biochemical functions
rounding the cytoplasm, in which a variety of (Table 1.1) and allow more ordered and direct-
structures may be present. Some of these ed metabolism to occur. In addition, multicel-
structures may themselves be bounded by lular eukaryotic organisms have evolved cells
membranes. In certain cells the plasma mem- with very specialized functions and structures
brane may be enclosed by a cell wall. which are often associated in large numbers to
Depending on the complexity of the internal form clearly identifiable tissues. The structure
structure, cells have been classified into two of typical animal and plant cells is shown in
basic types - prokaryotic and eukaryotic cells. Figure 1.1.
Cells that have no internal, membrane-
bounded structures and no clearly defined 1.2 COMPONENTS OF CELLS
nucleus are defined as prokaryotic cells.
1.2.1 PLASMA MEMBRANE
Bacteria and blue-green algae are examples of
prokaryotic cells. Although such cells have no The boundary of the cell is the plasma mem-
intracellular architecture, they contain all of brane. The composition, structure and func-
the metabolic machinery necessary to allow tion of plasma membranes are described in
them to grow and multiply. Chapter 22. The plasma membrane isolates the
Eukaryotic cells are much more complex internal contents of the cell from its environ-
than those of prokaryotes. Cells that contain a ment, and thus the cell is able to maintain a
nucleus surrounded by a membrane, the relatively ordered and constant environment
4 Cell structure and function
Table 1.1 Function of cell structures
Cell structure Function/pathway
Cell membrane Transport of nutrients; hormone/receptor interactions; cell recogni-

tion; permeability barrier
Cytoplasm Glycolysis; gluconeogenesis; pentose phosphate pathway; polysac-
charide breakdown; complex lipid breakdown; fatty acid synthesis;
protein breakdown; amino acid synthesis
Smooth endoplasmic reticulum Fatty acid elongation and desaturation; complex lipid synthesis;
detoxification reactions
Ribosomes Protein synthesis (proteins destined for storage or secretion are syn-
thesized by ribosomes attached to the endoplasmic reticulum)
Chloroplast Photosynthesis; fatty acid synthesis; complex lipid synthesis; synthe-
sis of some amino acids; synthesis of organelle proteins; Calvin cycle
(stroma); light reactions (thylakoids); reduction of nitrate and sul-
phate, part of photorespiration
Mitochondria - matrix TCA cycle; fatty acid oxidation; amino acid oxidation; gluconeogene-
sis; synthesis of organelle proteins
Inner mitochondrial membrane Electron transport chain; oxidative phosphorylation; metabolite
transport
Nucleus DNA synthesis; RNA synthesis; processing of RNA
Golgi bodies Carbohydrate synthesis (e.g. lactose); glycoprotein synthesis (addition
of sugar residues to existing proteins); packaging of cell products
Peroxisomes fatty acid oxidation; amino acid oxidation; photorespiration
Glyoxysomes Glyoxylate pathway; fatty acid oxidation; amino acid oxidation
Lysosomes Lipoprotein breakdown; recycling of cellular constituents; destruc-
tion of foreign bodies
Plant vacuoles Maintenance of turgor; storage of toxic and waste products
Cytoskeleton Cytoplasmic streaming; movement of subcellular organelles; mainte-
nance of cell shape
Flagella and cilia Movement
despite large changes in the composition of hydrophobic barrier that prevents the passage
the medium in which it lives and grows. This of most polar molecules such as inorganic ions,
is possible because the plasma membrane of all sugars and proteins. Some of the proteins
organisms is a selectively permeable barrier within the membranes have specific functions.
that controls the movement of molecules into They may act as transport proteins, enzymes,
and out of the cell. Essential nutrients required or recognition proteins such as receptors.
for growth and metabolism are allowed to These are discussed in more detail in later
cross the plasma membrane into the cell, often chapters.
by tightly controlled, energy-dependent trans-
port processes. Waste products produced by
1.2.2 CYTOPLASM
the cell, which if allowed to accumulate would
be toxic, are excreted by similar mechanisms. Inside the plasma membrane is the cytoplasm.
Although the plasma membranes of cells have This is composed of the cytosol, and the struc-
adapted with time to have specialized func- tures contained within it. The cytosol is an
tions, their essential feature is the presence of aqueous solution in which are dissolved many
a lipid bilayer, composed mainly of phospho- organic compounds such as sugars, amino
lipid and protein. This bilayer forms a acids, proteins and inorganic materials. In
Cilia
Peroxisome Vacuole
Chloroplast
Nucleus Smooth
endoplasmic
reticulum Smooth
endoplasmic
reticulum
Cell wall iii

Plasma
membrane
Wall of
Golgi adjoining
vesicles cell
membrane
vesicles
membranous
Rough endoplasmic Centrioles Rough endoplasmic Filamentous vesicles
Filamentous
reticulum reticulum cytoskeleton
cytoskeleton
(a) (b)
Figure!.! Structure of (a) an animal cell and (b) a plant cell. Not all of the intracellular organelles and plasma membrane-associated structures
are found in every cell: the numbers of some organelles and the complexity of the intracellular structure vary in cells according to their func-
tion. (Reproduced with permission from Darnell, J.E., Lodish, H.P. and Baltimore, D. (1990) Molecular Cell Biology, Scientific American Books
Inc.)
addition, the cytoplasm may contain insoluble no outer membrane. Bacteria with this type of
particles and organelles. In the cells of higher wall are stained by Gentian violet and are
organisms these structures can be numerous referred to as Gram positive.
and complex, but in bacteria the intracellular Plant cell walls consist mainly of cellulose
structure is quite simple. and other complex sugars. Because plants
have no skeleton, the rigidity provided by the
wall is important in providing support and
1.2.3 THE NUCLEOID AND NUCLEUS
protection to the plant and in enabling it to
The DNA in bacteria is in the form of a single grow upright and to reach a considerable
circular strand, tightly folded with protein to height.
form the nucleoid. Some bacteria also contain
circular DNA in the cytoplasm, referred to as
1.2.5 RIBOSOMES
plasmids.
The most noticeable feature of eukaryotic Ribosomes are responsible for the synthesis of
cells is the presence of a nucleus surrounded proteins. They are composed of two subunits,
by a double membrane known as the nuclear both of which contain RNA and protein. The
envelope. This envelope has holes or pores subunits of prokaryotic ribosomes are classi-
that allow communication and controlled fied as 50S and 30S in size and these combine
exchange of material between the cytoplasm to form a 70S ribosome. Eukaryotic ribosomes
and the nuclear contents. For example, the consist of 60S and 40S subunits which combine
messenger and ribosomal RNA synthesized in to form 80S ribosomes. An explanation of these
the nucleus is transferred to the cytoplasm terms is given in Chapter 21. In eukaryotes
where protein synthesis takes place. DNA con- some ribosomes may be attached to the mem-
tained in the nucleus forms a complex with branes of the endoplasmic reticulum.
proteins to give chromatin. The part of the
DNA required for the production of ribosomes
1.2.6 ENDOPLASMIC RETICULUM
is located in a dense area within the nucleus
known as the nucleolus. In eukaryotes the nuclear envelope is continu-
ous with a system of membranes in the cyto-
plasm called the endoplasmic reticulum (ER),
1.2.4 CELL WALLS
which is effectively a network of membrane-
The cells of plants and most prokaryotes are bounded tubes. There are two types of ER,
surrounded by a cell wall which is outside the rough and smooth.
cell membrane. The rough ER is the site of synthesis of pro-
There are essentially two different types of teins destined to be secreted from the cell
bacterial cell wall. One type consists of a thin (such as secretion of digestive enzymes or of
layer of peptidoglycan, a complex polymer of casein granules by the mammary tissue), or
sugars and amino acids which is surrounded which will become confined within lysosomes
by an outer membrane, similar in structure to or targeted to the nucleus. These proteins are
the plasma membrane, but with a somewhat synthesized on ribosomes attached to the ER
different composition. This type of cell wall membranes and are secreted into the lumen of
prevents the uptake of the dye Gentian violet, the ER where they can be directed to their spe-
first used by Gram as a means of identifying cific destinations. The 'rough' appearance of
bacteria (bacteria with this type of wall struc- this part of the ER is due to the presence of
ture are referred to as Gram-negative bacteria). ribosomes required for this process.
The other type of cell wall is composed of a The smooth ER is devoid of ribosomes but
much thicker layer of peptidoglycan and has is continuous with the rough ER. It is here
Components of cells 7
that the enzymes involved in complex lipid changes in the turgor of the cell, altering the
synthesis are located. In addition, the rigidity of plant tissues. The vacuole also
enzymes involved in detoxification of poten- serves as a site where materials may be stored,
tially harmful compounds are also found in separated from the main biochemical process-
the smooth ER. es of the cell. Thus pigments and toxic and
When cells are disrupted in the laboratory waste materials may accumulate in the vac-
in order to study the biochemical activity of uole. The presence of a vacuole also reduces
their subcellular components, the structure of the amount of cytoplasm required by plant
the ER is lost. The continuous membrane cells and allows them to reach a larger size
structure is broken into many small segments than most animal cells.
which form small vesicles referred to as micro- Lysosomes are spherical membrane-bound-
somes. These vesicles contain many of the ed vesicles found in the cytoplasm of animal
enzymes associated with the ER; these are cells. They are specialized types of vesicles
often referred to as microsomal enzymes, indi- produced by the Golgi bodies. Lysosomes con-
cating that they originated from the ER. tain hydrolytic enzymes such as the cathepsin
Eukaryotic cells contain specialized areas of proteases, lipases, nucleases and carbohy-
endoplasmic reticulum called Golgi bodies drate-degrading enzymes. The contents of the
(also called Golgi apparatus or dictyosomes). lysosome are more acidic than the cytoplasm,
Under the microscope the Golgi bodies appear pH 5.0 as compared to pH 7.0. The function of
to consist of layers of smooth ER stacked one the lysosome is to act as a focus for the recy-
on top of another. Each layer is referred to as a cling of redundant cellular components and in
cisternum. The ends of the cisternae give rise the digestion of foreign material, e.g. bacteria
to small vesicles which act as transport vesicles brought into the cell by phagocytosis. The pri-
and contain the products of biosynthetic mary lysosome, produced by the Golgi bodies,
processes which take place in the Golgi bodies. fuses with a vesicle containing a foreign body
These organelles are particularly numerous in or cell component (phagocytic or autophagic
secretory cells and it is clear that they are a vesicles) allowing the hydrolytic enzymes to
major site of packaging, modification and sort- degrade the contents to simple compounds
ing of cellular products. Many proteins are such as amino acids, sugars and fatty acids
modified in the Golgi bodies, for example by which are released into the cell for re-use.
the addition of sugar residues to form glyco- Several other types of vesicles are found in
proteins. animal and plant cells. Peroxisomes are the
Secretory vesicles produced by the Golgi site of specialized amino acid and fatty acid
bodies move to the cell membrane, where the degradation, which involves the production of
membrane surrounding the vesicle merges hydrogen peroxide and free radicals. Both of
with the cell membrane to discharge its con- these chemical species are highly reactive and
tents outside the cell. This process is called if released into the cytoplasm could cause
exocytosis or reverse pinocytosis. extensive damage. To prevent this happening
several antioxidant mechanisms are found in
peroxisomes. The first is the presence of the
1.2.7 VACUOLES AND SPECIALIZED VESICLES
enzyme catalase which degrades hydrogen
Plant cells contain vacuoles which are bound- peroxide to water and oxygen. The second is
ed by a single membrane known as the tono- the presence of the antioxidant vitamin E in
plast. In the young growing plant cell, several the peroxisomal membrane which reacts with
vacuoles may be found. These merge to form a free radicals to produce stable, less-reactive
larger vacuole as the cell matures. The move- compounds. Glyoxysomes are found only in
ment of water to and from the vacuole causes the cells of certain plants. They contain the
enzymes of the glyoxylate cycle (Chapter 11), a example those of the TCA cycle and fatty acid
specialized adaptation of the TCA cycle which oxidation. The number of mitochondria in
allows some plants to convert stored lipid into cells varies greatly. White adipose tissue, for
carbohydrate. They are usually found in seeds example, contains few mitochondria, where-
where significant quantities of fat can be as brown adipose tissue owes its colour to the
stored. Their numbers increase during seed large number of mitochondria it contains.
germination when the stored fat is mobilized Liver and muscle tissue contain many hun-
for sugar production. dreds of mitochondria per cell.
1.2.8 MITOCHONDRIA 1.2.9 CHLOROPLASTS
Both plant and animal cells contain mitochon- Chloroplasts are found in plant cells and share
dria. These subcellular organelles make up the some common features with mitochondria.
powerhouse of the cell. They are major sites of They are also organelles with a double mem-
ATP synthesis and oxidative metabolism brane structure and are involved in the trap-
(Chapters 11-13). In shape and dimensions ping of energy in the form of ATP. However,
they are similar to bacteria, and it is generally they are adapted to converting the energy in
agreed that mitochondria have evolved from sunlight into ATP, in contrast to mitochondria,
bacteria which developed a symbiotic relation- which use the chemical energy released dur-
ship with early eukaryotic cells. In support of ing the oxidation of organic compounds
this view is the observation that mitochondria (Chapter 17). The inner membrane of chloro-
contain DNA, RNA and ribosomes which plasts is highly folded to form numerous
resemble those in bacteria. Some mitochondri- stacks of membrane called thylakoids. Each
al proteins are synthesized in situ in the mito- fold, referred to as a thylakoid disc, contains
chondrial matrix, whereas others are pro- the units which carry out photosynthesis. The
duced in the cytoplasm from nuclear DNA ability to trap solar energy is conferred by the
(Chapter 21). presence of specialized pigments, mainly
Mitochondria have a double membrane chlorophylls a and b, in the inner membrane
structure. The outer membrane is relatively of the chloroplast. The aqueous contents of the
simple and unfolded, whereas the inner chloroplast are called the stroma. It is believed
membrane is highly folded forming cristae that chloroplasts may have evolved from an
which project into the centre of the mito- early form of cyanobacter which developed an
chondria. The outer membrane has limited endosymbiotic relationship with progenitor
biochemical activity and is permeable to most eukaryotic cell lines.
molecular species. Thus, the composition of Photosynthetic prokaryotes lack chloro-
the cytoplasm and the contents of the mito- plasts but are able to carry out photosynthesis
chondrial intermembrane space is similar. because they have pigments embedded in
The inner mitochondrial membrane has other specialized membranes.
many important biochemical functions, and
this is reflected in its composition which is
1.2.10 CYTOSKELETON
approximately 75% protein and 25% lipid. It
is the site of the mitochondrial electron trans- Many free-living cells are able to move in their
port chain and the synthesis of ATP by oxida- growth medium. In the case of bacteria and
tive phosphorylation (Chapter 12). The cen- protozoa this is due to the presence of special-
tral compartment of the mitochondrion is ized protein fibres called flagella which rotate
called the matrix. This is a concentrated aque- to propel the cell forwards. Other organisms
ous solution containing many enzymes, for such as amoeba are able to move without fla-
Cell specialization and interaction 9
gella and this movement is attributed to cyto- which confers the strength to hold a particular
plasmic streaming. shape and allows positioning of subcellular
Many eukaryotic cells, from simple unicel- organelles.
lular organisms to complex multicellular high-
er plants and animals, contain a cytoskeleton.
1.3 CELL SPECIALIZATION AND
This is a system of protein filaments and
INTERACTION
tubules distributed around the cell, which acts
not only as a mechanism for cell movement The size which individual cells can reach is
but also as a framework for the organisation, limited mainly by the ratio of their surface area
positioning and relocation of subcellular to volume. As cells become larger their volume
organelles, and provides strengthening to sta- increases in relation to their surface area, so
bilize cell shape. Although the nature of the that the speed with which nutrients and gases
cytoskeleton of eukaryotic cells is complex and diffuse from the cell membrane into all regions
diverse, three principal components have of the cell becomes limiting. Large cells usual-
been identified: actin filaments, micro tubules ly have some adaptation which allows them to
and intermediate fibres. cope with this situation. In many large plant
ACtin filaments are composed of thousands cells, for example, the cytoplasm is restricted
of units of the protein actin, which can poly- to a narrow layer inside the plasma membrane
merize and depolymerize as the cell's require- and they may have specialized structures to
ments change. Another protein, myosin, can increase cytoplasmic streaming. This also
bind to actin, and in the presence of ATP reduces the energy-requiring need to synthe-
myosin is able to move along the actin fila- size cytoplasm as much of the remainder of
ment. Both actin and myosin are also compo- the cell is occupied by the vacuole. Another
nents of muscle cells and are involved in mus- adaptation which increases the surface area-
cle contraction (Chapter 32). In the context of to-volume ratio includes changing shape: for
the cytoskeleton it is thought that myosin can example, neurones of higher animals are very
attach to subcellular organelles and move long thin cells, and ganglia and kidney cells
them around the cell along the framework of have a highly convoluted cell membrane.
actin filaments. This may be one mechanism Cells in multicellular organisms may
by which cytoplasmic streaming is achieved. become highly specialized in the processes
Microtubules are hollow tubular structures which they carry out. Thus the biochemical
composed of two protein components, a and 13- processes described in this book do not have
tubulin. Associated with these microtubules are equal prominence in all cells, and this is often
two other proteins, kinesin and dynein, which reflected in their structure. All cells must
are thought to act on actin filaments in a similar carry out basic biochemical processes
way to myosin: they may be anchored to sub- required for growth and division. To do this
cellular organelles and, in the presence of ATP, they must respire to produce energy, synthe-
can move along the microtubules. Microtubules size proteins and nucleic acids, and make
become prominent during mitosis when cell other cellular components as they grow. In
division requires that the replicated DNA is general, each prokaryotic cell carries out
separated into the two new cells. They also these processes independently of other cells.
appear to be important in giving support and In complex multicellular organisms such as
movement to cilia and flagella. higher animals and green plants, individual
Intermediate filaments are composed of cells have become restricted in their functions
keratins, proteins commonly found in hair, and may make a specialized contribution to
hoof and nails. They are thought to provide a the well-being of the organism as a whole.
framework under the cytoplasmic membrane Thus these organisms contain a variety of
specialized cell types which are normally The efficient operation of a multicellular
organised into organs. This process of spe- organism in which each part contributes to the
cialization of function in multicellular organ- whole requires complex control mechanisms
isms is called differentiation. by which the biochemical processes can be reg-
In animals and plants, therefore, individual ulated, and efficient systems by which cells can
organs are specialized in carrying out specific communicate with one another over both short
functions. Some of these, which will be and long distances. These control systems pre-
referred to frequently throughout the book, sent opportunities for the manipulation of pro-
are presented in Table 1.2. ductive processes in both animals and plants.
Table 1.2 Examples of tissue and organ specialization
Tissue/organ Specialization
Animals
Brain and nervous tissue Generation and transmission of nerve impulses requiring ion transport
across membranes by active transport - the ATP required is generated
by oxidation of glucose
Liver Processing and redirection of tissue metabolites: glycogen synthesis,
gluconeogenesis, fatty acid synthesis (main site in birds and some non-
ruminants), lipoprotein synthesis, cholesterol synthesis, synthesis of bile
salts
Adipose tissue Fat storage, triacylglycerol synthesis, lipolysis, fatty acid synthesis (main
site in ruminants), non-shivering thermogenesis (uncoupled fatty acid
oxidation in brown adipose tissue)
Muscle Glycogen synthesis and breakdown, movement -the ATP required is
generated by oxidation of glucose
Kidney Excretion of waste products and resorption of minerals, gluconeogene-
sis (approx. 10% of total)
Digestive tract Secretion of digestive enzymes requires high rates of protein synthesis,
absorption of end products of digestion, often by active transport - the
ATP reqUired is generated by oxidation of glucose
Mammary tissue Synthesis of protein, lactose, fatty acids and triacylglycerols; secretion of
milk
Bone Synthesis of collagen and formation of crystalline matrix of bone, provi-
sion of the rigid internal framework of the body, reserve of calcium and
phosphorus
Plants
Seeds Synthesis and breakdown of starch and triacylglycerols, synthesis of
sucrose, synthesis and oxidation of fatty acids, protein synthesis
Leaves and other green tissue Photosynthesis, photorespiration, protein synthesis, transpiration
Roots Uptake of nutrients and water from the soil, assimilation of nitrate,
nitrogen fixation
Tubers and storage organs Starch synthesis and remobilization
Fruits Synthesis and storage of sugars, respiration, cell wall degradation, syn-
theis and degradation of pigments, accumulation of fruit acids
WATER AND SOLUTIONS 2
2.1 Introduction 11
2.2 The ionization of water 12
2.2.1 The pH of water 12
2.3 What are acids and bases? 14
2.4 Biological systems, ionic strength and pH 14
2.4.1 Stabilization of pH by buffers 14
2.5 Colligative properties 15
2.5.1 Depression of freezing point 16
2.5.2 Osmotic pressure 16
2.5.3 Semipermeable membranes that allow 17
some solutes to pass
2.1 INTRODUCTION Water is a compound of hydrogen and

As a chemical compound water is somewhat oxygen, two atoms of hydrogen being joined
unusual. It has a molecular weight of just 18. to each one of oxygen. The bonding in water
Most chemical compounds of this size are not is covalent: each pair of electrons is not fixed
liquids at normal temperatures and pressures. in place, but follows a complex three-dimen-
For instance molecular oxygen, 02' has a mole- sional path which takes the electrons for part
cular weight of 32 and does not condense into of their time around the oxygen atom and the
a liquid until the temperature reaches -183° C. rest of the time around the hydrogen. When
Hydrogen sulphide (H2S) with a molecular the electrons are situated near the oxygen
weight of 34, nearly twice that of water, is a gas atom then all that is 'visible' of the hydrogen
at normal temperatures and pressures. Water is a positively charged nucleus. The part of
obviously has some peculiar properties, some- this orbital around the oxygen atom is much
times referred to as its anomalous properties. bigger than the part around the hydrogen, so
The puzzle becomes even stranger when we the electrons spend more of their time associ-
start to look at the way in which things dis- ated with the oxygen than with the hydro-
solve in water, and at the nature of solutions. gen. The result is that the hydrogen atoms, in
The difference between a true gas and a liq- the temporary absence of their electrons, take
uid is that in the gas the individual molecules on a small positive charge and the oxygen
(or atoms in a few cases) are not attracted to atom gains a slight negative one. The charges
one another or are only weakly attracted, and are quite small, but are big enough to have
so can freely move throughout all the space enormous effects on the properties of the
that is available to them. In a liquid there is water. A hydrogen atom in one water mole-
some attraction between individual molecules cule will have an attraction for the oxygen in
and the whole quantity sticks together. On the another - this pattern of attraction of one
other hand, this process of adhesion is not molecule for another has the effect of aggre-
strong enough to force the liquid to cohere gating the molecules. They are not free to
into a definite shape and, for this reason, liq- move as in a gas. Aggregation of molecules
uids can flow. caused by hydrogen atoms with small,
12 Water and solutions
unshielded, electrical charges is called hydro- 2.2.1 THE PH OF WATER
gen bonding and is very common in bio-
Water dissociates into hydrogen and hydroxyl
chemical systems.
ions according to the equation:
HzO ~ H+ + OH-
2.2 THE IONIZATION OF WATER For every chemical reaction that comes to
an equilibrium point we can calculate an equi-
Because water carries different amounts of
librium constant; for the equation above this is
electrical charge in different parts of the mole-
given as:
cule it is known as a polar chemical com-
pound. Another interesting property of water
is that in a small proportion of water mole-
cules the bond that holds one of the hydrogen
atoms breaks, this comes away from the rest of
Note that the figures in square brackets are
the molecule but minus its electron. The only
the concentrations.
part of a hydrogen atom that is left is the
Because the concentration of the water in
nucleus, which is a single positively charged
the form of H 20 is massive in comparison to
particle called a proton or hydrogen ion, H +.
the amounts of ionized water, we can neglect
(A tiny proportion of hydrogen atoms also
any changes in its concentration. A new con-
have a neutron in the nucleus but this can be
stant can be defined which is called the ionic
ignored for most purposes.)
product of the water:
Once the hydrogen ion has departed, the
part of the water that is left has an extra elec-
tron which gives it a negative charge, this is
At 25° C the concentration of H+ is 1 x
the hydroxyl ion, OR. For every proton that
1O-?M. In pure water the concentration of OR
is formed there must be a hydroxyl ion,
ions must be exactly the same. We can there-
which means that overall the electrical charge
fore calculate Kw as:
in the water is balanced. In pure water only
one water molecule in ten million is split in Kw = (1 x 10-7) x (1 x 10-7)
this way. In most liquids any ion that drifted
away from the rest of a molecule would soon
Kw =1X 10-14
be recaptured. The difference is that in water, At anyone temperature this figure is a con-
hydrogen bonding again comes into play. As stant, so that if for any reason the concentra-
soon as a hydrogen ion comes into existence tion of H+ ions increases then the concentra-
it is immediately surrounded by a layer of tion of OR ions will have to decrease.
water molecules all arranged with their nega- The concentration of H+ ions is a measure
tive charges (the ones on the oxygen atom) of the acidity of a solution. Unfortunately a
pointing towards the positive charge of the scale that uses figures such as 10-14 is not very
free hydrogen (Figure 2.1). The same thing useful in practice, so acidity is expressed as
happens to the free hydroxyl ion, although in pH, which is related to the hydrogen ion con-
this case the water molecules of the sur- centration by the formula:
rounding layer are aligned so that their posi-
tively charged hydrogen atoms point
pH = -loglO[H+]
towards the negative ion (Figure 2.1). These When the hydrogen ion concentration is
layers stabilize the ions so that they can exist exactly the same as the hydroxyl ion concen-
separately. tration ([H+] = [OH-] = 10-7), the pH equals 7,
The ionization of water 13
26-
26-
6+~6+
4-
6+~6+
~6- ~6-
".HYdrOXYI ion
, , + ./ /
. H (negative charge) '(G\ Hydrogen ion

~ (positive charge)
/ , / , ,26-
4A\
26- /
6+~~ 6+~6+
~6- ~6-
Figure 2.1 Hydroxyl (OH-) ions and hydrogen (H+) ions are stabilized by being surrounded by a layer of
water molecules held in place by electrical charges.
and this is referred to as a neutral solution. The organic acid, acetic acid, is a very good
Where the pH is lower than 7 there is an example of a compound which is soluble in
excess of hydrogen ions and the solution is water but which is only partially dissociated in
said to be acid. If the pH is greater than 7 the solution:
solution is alkaline. CH3.COOH :;:= CH3.COO- + H+
Water is a very good solvent for a whole
range of chemicals (solutes). It dissolves In a pure solution in water, only about one
solids such as common salt and sugar molecule in 250 would be dissociated. Acetic
acid is a weak acid because it does not supply
extremely well and it mixes freely with a
as many H+ ions as a strong acid such as HCl,
number of other liquids such as alcohol
which is completely dissociated. Organic acids
(ethanol). On the other hand there are many
such as acetic acid are usually present in bio-
substances that do not dissolve at all well in
logical solutions in the form of their salts and
water: fats and oils are obvious examples.
in this case would be completely dissociated.
Liquids such as kerosene or petroleum are
The formula for the acetate anion is CH3COO-
not miscible with water although they will
but in this book the formula CH3COOH is fre-
dissolve fats, oils and grease. In general, quently used for simplicity.
materials that dissolve in water are them- A similar situation exists with alkaline com-
selves polar compounds, whereas those that pounds. These supply hydroxyl, OH-, ions
are insoluble are non-polar. and consequently reduce the concentration of
Many of the compounds that dissolve in hydrogen ions in the solution. Some alkalis
water dissociate into ions in the water. The ions dissociate completely, good examples being
that are formed are stabilized by hydration the hydroxides of sodium or potassium.
using hydrogen bonds. Some compounds are
completely dissociated when they are in solu- NaOH :;:= Na+ + OH-
tion. For example common salt, NaCl, breaks These are the strong alkalis or strong bases.
down into the positively charged Na+ (a cation) On the other hand, weak alkalis or weak bases
and a negatively charged CI- ion (an anion). such as ammonium hydroxide are only par-
Other compounds dissociate only partially. tially dissociated in solution.
NHpH ~ NH4 + + OH- ionic strength or pH will lead to damage of the
very sensitive molecules that are responsible for
For any compound that is only partially dis- the metabolism of cells. In agriculture, animals
sociated into ions we can define a constant and plants manage to survive under conditions
that describes the proportion that is dissociat- which are not always helpful to maintaining
ed. constantly favourable conditions. Crop plants
For acetic acid: often suffer from periodic loss of water, and
even under mild drought conditions some still
manage to survive. Animals too can suffer from
water deprivation. Cattle in the hot tropics may
only have access to water every 3 days or so. In
For ammonium hydroxide: the interim they will lose large amounts of
water both by excretion and by the evaporation
necessary for them to keep cool. Some breeds of
cattle are capable of losing up to 25% of their
body weight in this way and of making good
Where Ka and Kb are the dissociation con- the losses within a few minutes when drinking
stants. water becomes available. The changes in the
amounts of water lead to enormous variations
2.3 WHAT ARE ACIDS AND BASES? in the strength of solutions both inside and out-
side cells. Despite all these changes, animals
So far we have used the terms acid and base and plants manage to survive them on a regu-
without defining them. As we move into bio- lar basis and even to thrive. Within biological
chemistry, it will be necessary to have a very systems there must be some way in which the
clear idea of what acids and bases are. One of properties of solutions are stabilized so as not to
the simplest and best working definitions is: damage the other constituents of cells. Much of
• an acid is a hydrogen ion (or proton) donor the stabilization comes from a process of buffer-
• a base is a hydrogen ion (or proton) acceptor. ing whereby some of the compounds in cells
are able to cushion the changes. The pH values
If we look again at the equation for the dis- for a number of biological materials are shown
sociation of acetic acid: in Table 2.1.
Acid Base 2.4.1 STABILIZATION OF PH BY BUFFERS

(donates a proton) (can accept a proton) Solutions of weak acids and their salts can act
By this definition; water itself is an acid to stabilize pH over a given range and are
because it can donate a proton and the therefore known as buffers. If small amounts
hydroxyl ion is a base because it accepts one. Table 2.1 pH values for some common biological
systems
Hp ~ H+ + OH-
Material pH
2.4 BIOLOGICAL SYSTEMS, IONIC
Blood plasma 7.4
STRENGTH AND PH
Milk (fresh) 6.9
As we shall see in the chapter on enzymes Egg white 8
(Chapter 6), it is extremely important that the Grass silage 3.8-4.8
Tomato juice 4.3
pH of biological environments is maintained
Soils (note extreme variability) 3-11
within very close limits. Any large changes in
Colligative properties 15
of either OH ions (from an alkali) or H+ ions pH = pKa
(from an acid) are added to these solutions
there will be only a small change in pH. In the pKa = -log (1.75 x 10-5)
absence of the weak acid and its salt the
change in pH would have been much greater. pH = pKa = 4.76
If we return to the dissociation of acetic
acid: Acetic acid is a good example to use in the
chemical laboratory but it is not commonly
used by biochemical systems. In practice, in
cells there is a whole range of compounds
which act as buffers and are able to stabilize
(This is known to have a value of 1.75 x 10-5 the pH close to neutral. Some of their Ka and
at 25° C.) pKa values are shown in Table 2.2.
We can rewrite the equation so that: One place where pH is very important is in
the rumen of animals such as the cow. This is
the first compartment of the stomach' and is
I
extremely important in the digestion of plant

material which is the main source of nutrition
for these animals. If the pH in the rumen
This can be converted to an equation for pH
drops too low then digestion of food will be
by taking the negative logarithms (base 10) for
halted and there may be serious effects on the
both sides:
health of the animal. Dairy cows are often fed
on diets containing large amounts of grain,
[CH 3.COO-] and these can lead to very low pH values in
pH=pKa x [CH 3.COOH] the rumen. Work in many countries has
shown that the addition of buffers to the diet
Note that pKa = -loglO(KJ can maintain pH at normal levels and allow
Example: if we have a solution which is half animals to thrive on diets that would other-
molar with respect to sodium acetate and to wise damage their health. The commonest
acetic acid then in solution we have: buffer added under these circumstances is
CH3COONa -> CH3COO- + Na+ (1) sodium bicarbonate.
CH3COOH ~ CH 3COO- + H+ (2)

2.5 COLLIGA TIVE PROPERTIES
The dissociation of the salt, sodium acetate,
will be complete in solution so that we can say There are a number of properties of solutions
that the concentration of acetate (CH 3COO-) that depend upon the strength of the solution
from Equation 1 will be 0.5 M. On the other expressed in terms of the number of particles
hand, the extent of dissociation of the acid is dissolved in a given volume of solution (or sol-
very small so that the concentration of vent). The word particles is used rather than
CH3COOH will be very little different from molecules, because many molecules such as
0.5 M. In other words: salts that dissolve in water will dissociate into
two or more particles. On the other hand,
[CH3.COO-] = 0.5 = 1 sugar molecules which cannot dissociate will
[CH3.COOH] 0.5 contribute just one particle. Two of these
properties, the depression of freezing point
and the loglo of this must be zero. and osmotic pressure, are extremely important
Therefore: in agriculture.
Table 2.2 Dissociation constants and pKa values for common organic acids
Acid Formula Dissociation constant pKa
Formic acid HCOOH 1.77 x 10-4 3.75

Acetic acid CH 3COOH 1.75 x 10-5 4.76
Propionic acid CH3CH 2COOH 1.34 x 10-5 4.87
Lactic acid CH 3CH(OH)COOH 1.39 x 10-4 3.86
2.5.1 DEPRESSION OF FREEZING POINT intermingling of the layers until eventually all
of the contents of the test tube are at the same
It is well known that solutions have a lower
concentration. If the solutions are of widely
freezing point than their pure solvents. In cool
different specific gravities the process may
climates in winter, salt is routinely spread on
take a long time but equilibrium will eventual-
roads and paths to melt ice and snow, pre-
ly be achieved.
venting accidents to vehicles and pedestrians.
If the two layers are separated by a solid
This property has more subtle applications in
and impermeable partition then no mixing
that many plants can survive without being
can take place. A semipermeable barrier will
frozen at temperatures below 0° C. The
allow some small molecules to pass through it
depression in freezing point is about 1.86° C
whilst retaining larger ones. Many will only
for every mole of particle dissolved. This is
let water through. Most semipermeable mate-
fairly simple for a sugar such as glucose (MW
rials allow the passage of particles only when
180). A solution of 180 g glucose per litre will
they are formed into a very thin barrier. For
have a freezing point of -1.86° C. For sodium
this reason they are often called semiperme-
chloride (MW 58.5) a solution of 58.5 g 1-1 will
yield 1 mole of Cl- ion (particle weight 35.5) able membranes.
The principles of osmotic pressure are illus-
and 1 mole of Na+ ion (particle weight 23). The
trated by Figure 2.2. This shows two compart-
depression of freezing point will therefore be
ments, one filled with water, the other filled
3.72° C (Le. 2 x 1.86° C).
with a solution, and separated by a semiper-
A practical application is to be found in the
meable membrane. Water molecules can pass
standard testing of milk for adulteration with
freely through the membrane in both direc-
water. Milk ought to have a freezing point of
tions. There is a tendency for the concentra-
between -0.54 and -0.59° C; if it freezes at
tions of solute to equalize across the membrane
-0.52° C or higher then it is likely that the milk
and so more molecules will pass from the pure
has been diluted with water. Freezing point
water side to the solution than in the opposite
depressions of other biological fluids are
direction, so that the solution increases in vol-
shown in Table 2.3.
ume. As the volume grows so the pressure
increases, until it becomes so great that more
2.5.2 OSMOTIC PRESSURE
water molecules cannot pass through the
The osmotic pressure of different parts of membrane and the flow ceases. In the system
plants and animals is essential to maintaining shown in Figure 2.2, the extra pressure will
their function. The principle of the phenome- push water up the left-hand tube until there is
non rests upon the fact that when solutions of a difference in the height (h) of the columns of
different concentrations are put together their water. The height of this column of water is the
concentrations will tend to equalize. Thus, if osmotic pressure of the solution, and can be
two solutions of differing strengths are care- expressed in any of the normal units of pres-
fully layered in a test tube, there will be a slow sure: atmospheres, mm of water, bar or Pascal.
Colligative properties 17
Measuring osmotic pressure in this way is a
useful concept in chemistry, but in practice the
r- solutions that are met in biological systems are
F
so complex that the equations do not have
much meaning. For instance, much of the
osmotic pressure of biological fluids comes
from proteins, and it is almost impossible to
r- determine how many of the charged groups
on the protein will be ionized at anyone time
"II and to what extent. This means that we cannot
II + OAlb calculate M with any degree of accuracy.
O,Hb -. II
For biological systems we use a unit called
II
II AI
osmolarity, which is the molarity of an ideal
11+ 0 b solution which exerts the same osmotic pres-
II sure as the test solution. Osmolarity is mea-
SOlution/" II H
Water sured in Osmoles (osm) or, more commonly,
II .... 0' b in the smaller milliosmoles (mosm). One inter-
II esting finding is that, in most mammalian sys-
/ tems, body fluids exert more or less the same

osmotic pressure (Table 2.3).
Cell membranes are semipermeable and the
Semipermeable membrane
cytoplasm inside has an osmotic pressure. If
Figure 2.2 Osmosis: water molecules pass through cells are surrounded by a fluid of a different
the semipermeable membrane in the direction of osmotic pressure there will be a flow of water
the solution in an attempt to balance concentra- from the region of low osmotic pressure to
tions on both sides of the membrane. The pressure that of high pressure. This is easily demon-
in the solution compartment increases until it strated by putting red blood cells into water:
reaches a point (height h) where it prevents fur- so much water flows into the cells that they
ther water from passing through the membrane.
simply burst. On the other hand, if the cells
are placed in strong salt solutions, water flows
The value of the osmotic pressure for an out of the cells so that they shrivel. Solutions
ideal solution (one in which the individual that have the same osmotic pressure as blood
molecules of solute have no effect on one are said to be isotonic.
another) is given by the equation:
B=MRT 2.5.3 SEMIPERMEABLE MEMBRANES THAT
ALLOW SOME SOLUTES TO PASS
Where B (the osmotic pressure in atmos-
pheres) is determined by M (the total molarity In looking at osmotic pressure we have
of the solution), R (the gas law constant) and T assumed that the only thing that could pass
(the absolute temperature in degrees Kelvin). through the membrane was water. In real bio-
In these units, R = 0.082l. logical systems, cell membranes do not behave
The depression of freezing point of a solu- like this - they allow some solutes (usually
tion can be used to calculate its osmotic pres- small molecules or ions) to pass, but others are
sure using the equation: retained (see Chapter 22). The problem may be
further complicated by the fact that the cell
B = 0.044IT.LlT
itself is able selectively to modify and regulate
where LlT is the depression of freezing point. the permeability of its plasma membrane.
Table 2.3 Colligative properties, depression of freezing point
and osmotic pressure in some biological fluids
Fluid Depression of freezing Osmotic pressure

point (OC) (mosm)
Blood -0.54 302

Cerebrospinal fluid -0.57 306
Milk -0.56 304
Semen -0.57 296
Consider Figure 2.3a: two compartments of as the Donnan equilibrium, and it has big
the same volume are separated by a semiper- implications for the behaviour of cells.
meable membrane that can allow the passage
of both K+ and Cl-. If water is placed in one (a)
side and 0.2 M KCI on the other, there will be
a flow of ions from one side to the other so as
to equalize the concentration. At equilibrium
there will be a 0.1 M solution of KCl on each
side. But if we place a solution that consists of
0.1 M KCl on either side but in addition one
has 0.05 M KR as well (where R' is a cation too
big to pass through the membrane) the system
cannot come to equilibrium with the same
concentrations on each side of the membrane
(Figure 2.3b).
On either side of the membrane, the K+
ions must balance the total of negative ions. In
the system described we will finish up with (b)
the conditions shown in Table 2.4.
This means that the concentration of K+ and
Cl- is different on each side of the membrane; it
:!Frcr :: hr:! K~cr !:: ·::::::::::cr::::::::
.~.~:~~.~'~'~.~:~:~ -~:~:~-.-'-~:~:~~
also means that the total electrical charges are

not the same on the two sides (Figure 2.3b).
:~~ :':::::::: :::c(
This situation is an example of what is known
.::::::~::::: .
::::::i<+.....
.....
'....... ,.
Table 2.4 Concentrations of solutes after equi-
::::;C(:
librium is attained
.:::: ....... :C.I"::::::::::::
Element Left-hand side Right-hand side
Figure 2.3 Donnan equilibrium. (a) The membrane
K+ 0.139 0.111 is freely permeable to both K+ and Cl- ions and the
Cl- 0.089 0.111 concentration of each will equalize across the
R- 0.05 o membrane. (b) The membrane is not permeable to
CI- + R- 0.139 0.111 R- ions and thus an imbalance of concentration
and charge will result.
THE CARBOHYDRATES 3
3.1 Introduction 19
3.2 Structures of sugars 20
3.2.1 Optical isomers 20
3.3 Naming of sugars 20
3.4 Sugars with four carbon atoms, the 20
tetroses
3.5 Sugars with five carbon atoms, the 21
pentoses
3.5.1 Ring formation in sugars 22
3.5.2 Five- and six-membered rings 22
3.5.3 Ring formation is not permanent 23
3.6 Sugars with six carbon atoms, the 23
hexoses
3.6.1 Glucose 23
3.6.2 Fructose 23
3.6.3 Other hexoses 23
3.7 Reducing and non-reducing sugars 24
3.8 Formation of sugar acetals 24
3.8.1 Formation of disaccharides 25
3.8.2 Sucrose 25
3.9 Polysaccharides 26
3.9.1 The storage carbohydrates - starch 26
and glycogen
3.9.2 Structural polysaccharides in plants 29
3.9.3 Other polysaccharides and related 32
compounds
3.1 INTRODUCTION simple sugars were all found to have the

The carbohydrates are a group of organic empirical formula CIIHzIIO II , and for this rea-
compounds that includes sugars and related son they were assumed to have arisen by
compounds. The sugars are compounds with some form of hydration of carbon (i.e. C + II
between 3 and 7 carbon atoms having many nHzO). The name carbohydrates has stayed
hydroxyl (alcohol) groups and either a ketone with them long after their real structures were
group or an aldehyde group. Typically, each discovered.
carbon atom that does not bear an aldehyde Cells of all types use sugars as a convenient
or ketone group (collectively called carbonyl source of energy and as the raw materials for
groups) will have a hydroxyl group. Simple many chemical syntheses. The biochemical
sugars may be strung together in chains to pathways for making sugars are relatively sim-
form long polymers. When they were first ple, and many cells, both in animals and
carefully investigated, in the 19th century, the plants, make huge quantities of them. They
20 The carbohydrates
are water soluble and are therefore easily deviate the beam of polarized light to the left
transported either in blood or phloem. and the other to the right. They have the same
formula so they are isomers, and because they
differ in the effect that they have on light they
3.2 STRUCTURES OF SUGARS
are known as optical isomers. The carbon atom
The simplest sugars contain just three carbon that can be bonded in either of two ways is
atoms. Two different formulae are possible called the optical centre and is said to be opti-
(Figure 3.1a) depending upon whether the cally active or asymmetric.
sugar has a ketone or an aldehyde group. Although the structural formulae appear
Although these are the simplest of all the sug- very similar on paper, in real biological sys-
ars, they are not commonly found in these tems these two compounds will behave quite
forms but occur as their phosphate esters, as differently. Almost all carbohydrates exist as
intermediates in metabolic pathways. The structural and optical isomers, which leads to a
structure of the one with the ketone group, fascinating diversity in the properties of these
dihydroxyacetone, is unique but when we compounds.
look at the aldehyde sugar, glyceraldehyde, a
new level of complexity emerges in the form
3.3 NAMING OF SUGARS
of optical isomers.
The chemical names of the sugars and many of
the more complex carbohydrates end with the
3.2.1 OPTICAL ISOMERS
suffix -ose. They are also named on a basis of
Chemically the element carbon is known as the number of carbon atoms that they contain;
tetravalent, which means that each carbon tri- for three, and tetra-, pent-, hex-, and hept-
atom is joined to neighbouring atoms by four for 4, 5, 6, and 7, respectively. The type of car-
bonds. If all of are these are single bonds then bonyl group is denoted by the prefix of aldo-
they are arranged around the carbon atom in a for an aldehyde and keto- for a ketone. For
three-dimensional tetrahedral shape. A example, glyceraldehyde is an aldo-triose.
strange thing happens if one particular carbon
atom is joined to four atoms or groups which
3.4 SUGARS WITH FOUR CARBON ATOMS,
are themselves different: it becomes possible
THE TETROSES
to arrange the surrounding atoms or groups in
either of two different ways (Figure 3.2). On Four different aldo-tetroses are possible
paper they look similar but in biological terms because there are two different asymmetric
they may be quite different. Take for example carbons in the chain (see Figure 3.1b). In the
the simplest of the aldehyde sugars, glycer- natural world, the tetroses are quite rare in
aldehyde. The two carbons at the end of the their free form although several do appear as
chain do not have a chance to show this prop- intermediates in metabolic processes.
erty; one has two identical hydrogen atoms The carbon at the bottom of each structure
attached and the other has two of its bonds as is, by convention, the one with the highest
a double bond attached to the same oxygen number - in this case, 4. This carbon atom is
atom. The middle carbon atom (marked with always a CH20H group. Note that two of the
an asterisk) is attached to four different sugars are referred to as 'L' sugars and two are
groups; -H, -OH, CHpH and -CHao The two 'D'. All sugars (with the exception of dihy-
compounds in Figure 3.2 are actually mirror droxyacetone) are divided into these two
images of one another. Their solutions in groups depending upon the particular optical
water also differ in the effect that they have on isomerization at the carbon next to the CHzOH
polarized light as it passes through: one will group. By convention the hydrogen atom on
Sugars with five carbon atoms, the pentases 21
a. Triases
H'C-O CH 2 0H
I
'CH-OH
I
6-0
I
'CH 2 0H CH 2 0H
Glyceraldehyde Oihydroxyacetone
b. Tetrases
H'C-O HC-O HC-O HC-O
.1 I I I
CH-OH HO-CH HO-CH CH-OH
I I
'CH-OH CH-OH HO-CH HO-CH
• I I I I
CH 2 0H CH 2 0H CH 2 0H CH 2 0H
O-erythrose O-threose L-erythrose L-threose
C. Pentases
,
HC=O HC-O HC-O
.1 I I
CH-OH CH-OH CH-OH
.1 I I
HO-CH CH-OH HO-9H
.1 I
HO-CH CH-OH CH-OH
I I I
bH 2 0H CH 2 0H CH 2 0H
L-arabinose D-ribose D-xylose
o OH
~
.~-~, OH
HOf;~ HO OH
OH OH
Figure 3.1 Structures of some sugars with three, four or five carbon atoms. (a) Trioses: glyceraldehyde
exists as two different optical isomers (see Figure 3.2). (b) Tetroses: the tetroses shown are all aldo sugars,
the difference between them lies in the optical isomerization at positions 2 and 3. (c) Pentoses: these can
exist as straight chain sugars or in rings made by the formation of hemiacetal bonds. These are three of
the commonest pentoses found in agricultural materials.
this penultimate carbon is drawn pointing to isomers, the only L sugar normally encoun-
the left on an L sugar and to the right on a 0 tered is L-arabinose, one of the components of
sugar. Almost all of the natural sugars are 0 some complex structural carbohydrates in
plants.
a. b.
HO, ~o HO, ~O
c~ c~ 3.5 SUGARS WITH FIVE CARBON ATOMS,
I, I,
THE PENTOSES
/~CH OH HO/~CHOH
H HO H
2 2 Both ketoses and aldoses can be extended by
Figure 3.2 The two optical isomers of glyceralde- the addition of a further carbon atom and
hydroxyl group to give pentoses. This intro-
*
hyde are mirror images of one another. The car-
bon atom marked is asymmetrical. duces yet another asymmetric carbon atom
into each type of sugar and thus multiplies the cule between the carbonyl group and one of
number of possible isomers by two. Hence the alcohol groups further down the chain.
there are four possible keto-pentoses and eight This results in the straight chain of the sugar
aldo-pentoses. The aldo-pentoses are much being bent round on itself to form a ring which
more common in nature than the keto ones. also includes an oxygen atom (for instance, see
Before looking at the individual pentoses it is Figure 3.1c).
necessary to understand some of the chem- Once a hemiacetal has been formed, it can
istry of these compounds, because the added react with a further alcohol group to form an
chain length brings with it the possibility of acetal. During this reaction, water is given off (it
the straight chain looping round on itself to is therefore called a condensation reaction) and
form a ring. the process is not easily reversed. In the sugars
the formation of acetals involves a reaction
between the hemiacetal group on one sugar
3.5.1 RING FORMATION IN SUGARS
molecule and an alcohol from another. In this
As described above, sugars contain both alco- way two sugar molecules are linked together to
hol and carbonyl (aldehyde and ketone) form new and more complex carbohydrates.
groups. Many of the properties of the carbo- There is another consequence of the forma-
hydrates depend on addition reactions tion of hemiacetals: the carbon of an aldehyde
between these groups. The reactions can take is not asymmetric but that of a hemiacetal is, so
place between any alcohol and any carbonyl that there are two possible optical isomers of
group. Figure 3.3 shows an alcohol reacting each sugar hemiacetal.
with the carbonyl carbon atom of an aldehyde
to form a new compound called a hemiacetal.
3.5.2 FlVE- AND SIX-MEMBERED RINGS
This reaction is easily reversed and, if an alde-
hyde and alcohol are both dissolved in water, Three examples of aldo pentoses are illustrated
the solution will usually contain a mixture of in Figure 3.1c. Arabinose, ribose and xylose are
free aldehyde and alcohol molecules together quite common constituents of carbohydrates.
with hemiacetals. A similar reaction takes All have five carbon atoms induding the alde-
place between ketones and alcohols to form hyde group but the orientation (optical isomer-
hemiketals. In the sugars, hemiacetals (or ization) of the carbon in the middle of the
hemiketals) are formed within the same mole- chain is different, which means that the
H OH
=0
I
R-OH + R'-C
I
....---- R'- CI - H
------"'- .... 1
any any O-R
alcohol aldehyde
a hemiacetal
--
OHI
O-R .... 2
R'- C - H
I
R'- C - H
R-OH + I I + H2 O
O-R O-R
alcohol hemiacetal an acetal
Figure 3.3 The formation of hemiacetals (1) and of acetals (2). These reactions occur between aldehyde
and alcohol groups within the same sugar molecule.
Sugars with six carbon atoms, the hexoses 23
hydroxyls on these carbons stick out in oppo- 3.6.1 GLUCOSE
site directions. These small differences affect
The formation of the ring in glucose links
the way in which each of these molecules is
together carbons 1 and 5 of glucose through
formed into a ring. In arabinose and ribose the
the atom of oxygen and in doing so makes
hydroxyl of carbon 4 reacts with carbon 1 (the
carbon 1 asymmetric (Figure 3.4). The two
aldehyde) to form a five-membered ring.
different compounds are denoted as a- and
Carbon 5 sticks out from the ring. The five-
f3-glucose. In the straight-chain form of glu-
membered ring is called furan. In the case of
cose, carbon 1 is not asymmetric because it
xylose the hydroxyl on carbon 5 reacts with the
has two of its bonds attached by a double
aldehyde to give a six-membered, pyran, ring.
bond to an atom of oxygen. In ring-shaped
glucose, this carbon is effectively joined to
3.5.3 RING FORMATION IS NOT PERMANENT four different groups. The important differ-
ence to notice is that for a-glucose we draw
When sugars are in water solution, part will be
the hydroxyl group on carbon 1 as pointing
in the straight-chain form and the rest in the
downwards, whereas in the f3 form it points
ring form. If some process removes all the
up. These two versions of glucose are actual-
straight-chained sugar molecules from the
ly quite different compounds which do not
solution then some of the ring structures will
even have the same melting point. When glu-
open in order to replace the lost straight
cose is dissolved in water there is a rapid
chains and to restore the balance.
equilibrium between the three forms:
f3-D-glucose ~Straight chain glucose ~
3.6 SUGARS WITH SIX CARBON ATOMS, THE
a-D-glucose
HEXOSES
In solution at anyone time, most of the glu-
These sugars are the most common, indeed
cose is in the a form (65%) with smaller
their polymers probably account for a large
amounts as f3-ring (32%) and straight-chain
proportion of the solid carbon in the earth's
(3%) forms.
biosphere. They have one more asymmetric
carbon atom than the pentoses and thus eight 3.6.2 FRUCTOSE
keto-hexoses and 16 aldo-hexoses are possible.
Luckily there are only a few which are very This is probably the commonest keto-sugar
common in nature, and of these one aldose found (Figure 3.4). The commonest natural
(glucose) and one ketose (fructose) are partic- source of free fructose is honey, and this
ularly abundant. Like the pentoses, they form accounts for its very sweet flavour. Fructose is
either internal hemiacetals or hemiketals as quite commonly encountered as a component
appropriate. Theoretically, these can be of more complex carbohydrates, and it accounts
formed between the carbonyl group and any for half of the commercial sugar, sucrose.
of the alcohol groups, but in practice they only
3.6.3 OTHER HEXOSES
form between carbons 1 and 5 in the case of
the aldo-hexoses, and between carbons 2 and The ring forms for several other common
5 in the case of the keto-hexoses. The forma- hexoses are shown in Figure 3.5. Many of
tion of an internal hemiacetal or hemiketal these do not commonly occur as pure com-
makes the whole molecule of the sugar bend pounds but are to be found in combination
into the ring shape. In the case of glucose, the with other sugars. Figure 3.5 also shows the
ring which is formed is six-membered (pyran) structures of the related sugar acids, galactur-
whereas in the case of fructose it is five-mem- onic acid and glucuronic acid, which occur in
bered (furan). complex carbohydrates.
1
6 6"
6 HC-O
1
2 CH-OH
31
4 OH 1 HO-CH OH
1
4 CH-OH
1
HO 3 2 OH 5CH-OH HO
OH 1 OH
6 CH 2 0H
O!-D-glucose {3-D-glucose
1
6 1
9H 2 0H
"O~=,O" 2C-O
"O~"
HO~CH
1
54 3 OH 4CH-OH CH 2 0H
1
OH 5CH-OH OH
1
6CH 2 OH
O! -D-fructose {3 -D-fructose
Figure 3.4 The structures of glucose and fructose. In addition to the straight-chain form, each of these
sugars can exist in either of two ring structures, differing in the orientation of the carbonyl carbon (car-
bon 1 in glucose, carbon 2 in fructose) when the ring is formed.
3.7 REDUCING AND NON-REDUCING open-chain version of the sugar ready to take
SUGARS part in oxidation/reduction reactions.
True sugars all contain a carbonyl group,
either an aldehyde or a ketone. Both of these 3.8 FORMATION OF SUGAR ACETALS
are capable of being oxidized. In the case of
aldehyde groups, the product of oxidation is Acetals can be formed between any hemiacetal
the corresponding carboxylic acid. The and any alcohol group. Wherever there are
ketones are not as reactive and yield a whole sugars there are plenty of 'spare' alcohol
series of compounds. These sugars are there- groups, but in fact natural sugar acetals tend to
fore reducing agents. Many of the chemical be very specific. In the aldo-hexoses such as
methods used for detecting and measuring glucose, the hemiacetal is always formed at the
sugars depend on their reducing properties. In number 1 carbon atom so that this is always
alkaline solution, copper (II) sulphate will oxi- involved in acetal formation. The free hydrox-
dize an aldehyde to its corresponding car- yl group used in acetal formation is almost
boxylic acid leading to the formation of copper always the one on either carbon 4 or carbon 6
(I) oxide. The liquid changes from a bright blue of another sugar molecule.
solution to a suspension of a dark red precipi- Once an acetal has been formed it is quite
tate. This provides the standard method for difficult to break it. As long as this acetal bond
measuring sugars in fruits. remains intact then the hemiacetal formed at
Sugars can act as reducing agents only carbon number 1 cannot be disrupted.
whilst the carbonyl carbon atom is not firmly Once an acetal has been formed the ring
linked to any other molecule. In sugars the structure of the sugar is 'locked' - the ring can-
open-chain form has a free carbonyl group. If not be opened and must remain in either its a
this is oxidized then any hemiacetal or or 13 form. This locking of the carbonyl group
hemiketal rings can open to yield more of the of one of the sugars means that the group is
Formation of sugar aceta Is 25
HO6 OH
Glucose
OH
OH
H~ OH
OH
OH
Galactose
HO5 OH
Mannose
0
OH
6
COOH COOH
H~ OH
OH
OH HO~ OH
OH
OH HO
OH
H3C
NH
OH
-c-o
I
G alactu ron ic Glucuronic

N-acetyl-
acid acid
glucosamine
Figure 3.5 Some common hexose sugars and related compounds: glucose, galactose, mannose, galactur-
onic acid, glucuronic acid, N-acetylglucosamine. The acid and N-acetyl derivatives are found in complex
carbohydrates.
unable to take part in any oxidation/reduction fixed, the right-hand one is free to open to the
reactions. straight-chain form.
This means that in maltose, cellobiose and
lactose the aldehyde group of the left-hand
3.8.1 FORMATION OF DISACCHARIDES glucose is unable to take part in any oxida-
tion/reduction reactions. However, the right-
Two molecules of a simple sugar linked
hand glucose group can react in this way, so
together as an acetal are known as a disaccha-
these disaccharides still have reducing proper-
ride. The disaccharide with a bond between
ties.
the 1 carbon of an a-glucose and the 4 carbon
The monosaccharide components of carbo-
of another a-glucose is called maltose (Figure
hydrates are sometimes distinguished by
3.6a). The bond is called an a-1,4 glycosidic
referring to them as the non-reducing or the
link. If the left-hand sugar had been in the ~
reducing end of the chain. Thus in the dia-
form before linking then the compound
grams all of the left-hand glucose groups are
would be a ~-linked disaccharide. The com-
non-reducing, and the right-hand ones
pound of this sort which is comparable to mal-
reducing.
tose is called cellobiose. Lactose, the sugar
found in milk, resembles cellobiose but the
3.8.2 SUCROSE
left-hand sugar is galactose instead of glucose.
All these disaccharides are formed between The commonest disaccharide of commerce is
the hemiacetal carbon of the left-hand sugar sucrose, a compound of glucose (in the a ori-
and the 4 carbon of the right-hand one. Thus, entation) and fructose (in the ~ form).
whilst the ring shape of the left-hand sugar is Unusually, it is an acetal formed between the
cose unit of this new compound is free to join
with a further glucose unit, in which case it
would form a trisaccharide. The process can be
repeated many times to form long chains of
the simple sugars, the polysaccharides. In
most polysaccharide chains only the single
a. Maltose
sugar group at one end of the molecule is free
to open and express chemical reducing prop-
erties. The chains of a polysaccharide thus
carry both reducing and non-reducing ends.
Glucose is the commonest sugar in poly-
saccharides and it forms two main families of
carbohydrates which differ simply in
b. Cellobiose whether the units from which they are made
are in the a or 13 form. In general, those that
H~O~"J' ~O~"
are made up of l3-glucose units are physically
much stronger, are less soluble in water and
are much more difficult for animals to digest.
~O~OH Many of the common polysaccharides con-
OH OH tain only one type of sugar group and are
c. Lactose termed homopolysaccharides. Those with
two or more different sugars are het-
eropolysaccharides.
Almost all of the carbohydrates that are
commonly found in agriculture come from
plants, and here polysaccharides have two
main functions: storage and structure.
d. Sucrose
Figure 3.6 Common disaccharides: maltose, cel-
lobiose, lactose, sucrose. With the exception of 3.9.1 THE STORAGE CARBOHYDRATES-
sucrose, the ring of the right-hand glucose unit can STARCH AND GLYCOGEN
open exposing a free aldehyde group and giving
reducing properties to the sugar. Plants, and to a lesser extent animals, use car-
bohydrates as a way of storing nutrients for
future need. During the normal processes of
hemiacetal carbon of glucose and the hydrox- growth and regeneration, the plant stores
yl group of the hemiacetal in fructose. This materials in both seeds and roots to cover peri-
means that neither of the rings in sucrose can ods when its ability to supply nutrients from
open to the straight-chain form and therefore photosynthesis is inadequate. As these materi-
it is a non-reducing sugar. Huge quantities of als have to be broken down by the plant they
this sugar are produced either from sugarcane are not physically strong, nor do they have to
in tropical and semi-tropical areas or from be water-resistant. They are mainly polysac-
sugar beet in temperate zones. charides made up of glucose units in the a
form. The commonest examples are the starch-
es and starch-like materials that come from
3.9 POLYSACCHARIDES
seeds such as corn, and tubers such as pota-
Looking at the structures of maltose or cel- toes, yams and cassava. Unlike the structural
lobiose, it is quite clear that the right-hand glu- polysaccharides, animals digest them very eas-
Polysaccharides 27
ily and they form the staple ingredient of the The long chains of amylose roll themselves
human diet in most parts of the world. into a stable helix shape which is held in place
by hydrogen bonding (Figure 3.8). The helix is
a tube into which other molecules or atoms
Starch
can fit. One example of this is the fact that
Starch consists of a mixture of two different iodine can fit inside the helix and form a blue-
types of molecules: amylose, which is a long coloured complex with the amylose, a reaction
chain of glucose atoms joined by a-l,4 link- which is often used to detect the presence of
ages; and amylopectin, which consists of a either starch or iodine.
mixture of a-l,4 links with occasional a-l,6
branches. The branches occur after about 25
Glycogen
straight a-l,4 bonds (Figure 3.7). Starches
from different sources vary in the ratio of Animals also make use of an a-linked polysac-
amylose and amylopectin, in the size of the charide for storage of energy, although the
individual molecules, and in the degree of amount of material stored is very small. The
cross-linking in the amylopectin molecule. In compound used is called glycogen and is very
general, amylopectin accounts for about 70% similar in structure to amylopectin although
of starch. the molecules are larger and the cross-linkages
Figure 3.7 The structure of amylopectin, one of the components of starch. Chains of glucose groups,
linked a-l,4, contain occasional a-l,6 bonds that provide branching points. In native starches the branch-
ing points occur approximately every 25 glucose residues.
,,
\ I
o
OH
I
01)- , I
\ 0
/-(0 y.0 \
HO \ y.0
1-\0 0 \
HO 1-\0 0 Y'
o 0
"1-\') 0 o
HOGH,) 1-\0 I I
'"o
I
0
I
0
CH 2 0H
0
I
'"0
I
0
\ I
0
OH 0
01)-
0Qod
0
,
\
\
\
\
HOGH,) 1-\0"1-\') \
\
Figure 3.8 The structure of amylose, the second component of starch. Molecules consist of long chains of
glucose groups, linked a-l,4. These are held in their helical form by hydrogen bonding to form long
tubes.
Polysaccharides 29
more frequent (once every 15 or so straight residues covalently linked by J3-1,4-g1ycosidic
bonds). bonds. Long, linear chains of pure glucose are
termed glucans (Figure 3.9). Between 2000 and
6000 residues are polymerized in each glue an
3.9.2 STRUCTURAL POLYSACCHARIDES IN
chain of the primary wall. A single cellulose
PLANTS
fibre of 3.5-4 nm in diameter contains 30-40
In agriculture the non-starch polysaccha- glucan chains held together by a very large
rides can be viewed in two distinct ways. To number of hydrogen bonds. Within the fibre,
the crop scientist they are the materials from individual glucan chains are very much short-
which plants fashion much of the physical er than the length of the fibre; they overlap at
components of their structures. The basic units random and probably all have the same polar-
of the plant are its cells, and their structural ity, i.e. the reducing groups are all at the same
integrity comes from the materials that make end.
up the cell wall. On the other hand, the animal
scientist regards non-starch polysaccharides as
Hemicellulose
the materials that form the fibre components
of the diets eaten by livestock. This has led to In dicotyledonous plants the main hemicellu-
the development of a number of different loses are xyloglucans, mixed polysaccharides
terms for what are essentially the same materi- principally of glucose and xylose. Some xylan,
als: fibre carbohydrates, structural carbohy- a pure polymer of xylose, is also present. In
drates and cell-wall carbohydrates. addition to the major components of xylose
At least 90% of the structural material of cell and glucose, xyloglucan also contains fucose,
walls of all higher plants is polysaccharide. galactose and small amounts of arabinose. The
The remaining 10% is made up of protein xyloglucan has a J3-g1ucan backbone very like
which, like some structural proteins of animal that of cellulose and this can probably interact
cells, is rich in hydroxyproline. very strongly with cellulose by hydrogen
The main polysaccharides are cellulose, bonding (Figure 3.lOa). Fixed to about 75% of
hemicellulose and pectin. Originally these the glucose units of the glucan backbone are
terms referred to their relative solubilities in side chains of a-xylose. The xylose is a-l,6
strong acids and alkalis, however they are linked. A minority of the xylose units may also
now used to describe their molecular struc- bear another sugar such as galactose attached
tures as carbohydrates. In addition to the car- by a-l,4 bonds.
bohydrates, phenolic materials called lignins In monocotyledons and in legume leaves
are also present in some cell walls. and stems, the main hemicelluloses are arabi-
noxylans which consist of J3-1,4-linked xylose
residues with side chains at various points, but
Cellulose
some xyloglucan is also present (see Figure
Cellulose fibres make up the main structure of 3.lOb). Single arabinose units are the most
the cell wall and give the wall much of its common but arabino-xylose and arabino-xylo-
strength. The wall consists of cellulose rods or galactose side chains are also found. The poly-
microfibrils embedded in an amorphous meric xylan backbone serves the same func-
matrix of non-cellulose polysaccharides (hemi- tion as the xyloglucan in dicotyledons, in that
celluloses and pectins). Chemically the cellu- it can attach itself to cellulose through hydro-
lose consists of long, linear chains of glucose gen bonds.
,[QoJi?oJi?oJi?ii:;oJi?oJi?/Q/Q/Qii:;ii;i;_
OH OH OH OH OH OH OH OH OH OH OH OH
,[QoJi?oJi?oJi?oJi?oJi?oJi?oJi?/Q/QoJi?/Qi;_
OH OH OH OH OH OH OH OH OH OH OH OH
,[QoJi?oJi?oJi?oJi?/Q/Q/Q/Q/Q/Q/Qi;_
00 00 00 00 00 00 00 00 00 00 00 00
Figure 3.9 The structure of cellulose. Long chains of glucose groups, linked 13-1,4, associate by hydrogen bonding to form sheets. Adjacent
chains may lie parallel (as here) or may alternate in direction.
Polysaccharides 31
(a)
(b)
(c)
(d)
Figure 3.10 Structures of hemicelluloses and pectins. (a) The xyloglucans of dicotyledonous plants have a
backbone of glucose units with side chains of xylose. (b) Monocotyledons and legumes possess arabi-
noxylans which have a principal chain of xylose residues substituted with arabinose and other sugars. (c)
Galactosan pectins have a spine of f3-1,4-linked galactose units with arabinose branches. (d) The rhamno-
galactonuran pectins have chains of galacturonic acid, broken every 10 or so residues by a rhamnose
group.
Pectins degree of methylation of the COOH groups in
the pectin.
The main feature of the pectins is the presence
The commercial importance of pectins
of linear chains of galacturonic acid residues
results from their ability to form gels. Aqueous
(polygalacturonans), some of which are pre-
solutions of pectin, heated with sugar under
sent in the form of methyl esters. The poly-
acidic conditions (pH 2-3.5) solidifies to a clear
galacturonans also usually contain other sug-
gel on cooling and this forms the basis of jam-
ars, e.g. rhamnose (rhamnogalacturonans).
making.
Pectins are generally the most soluble of the
cell wall components. They are easily extract-
ed using hot water and they have physical 3.9.3 OTHER POLYSACCHARIDES AND
properties which make them important in the RELATED COMPOUNDS
food industry.
Saponins
The polygalacturonans may also be associ-
ated by covalent bonds to neutral pectic poly- Many forage legumes grown in temperate cli-
saccharides such as the arabinans, galactans mates contain saponins. These are glycosides
and arabinogalactans. consisting of a non-polar aglycone and a polar
The most common types of pectic polysac- sugar group. The non-polar group is either a
charides are indicated below. steroid or a similar polycyclic compound. They
therefore have strong detergent properties.
Saponins in alfalfa (lucerne) have been studied
Galactans and arabinogalactans
in detail. They are bitter and reduce intake
These arabinose- and galactose-containing when alfalfa is added to diets for non-rumi-
neutral pectic polysaccharides are thought to nant animals.
consist of homo-I3-1,4-linked galactose units Saponins form complexes with cholesterol
with few side chains linked to an arabinose and can reduce serum cholesterol levels
polymer with more arabinose branches (Figure because they prevent readsorption from bile.
3.10c). Pectin occurs in the primary and sec-
ondary cell walls and in the middle lamella.
Glucosinolates
Oilseed rape (canola) is widely grown as a
Rhamnogalacturonans
source of oil and the residue may be used as a
Pure polygalacturonans are quite rare - usual- feed component. However it contains types of
ly chains of polygalacturonic acid residues are compound which restrict its use: the glucosi-
broken by neutral rhamnose residues (Figure nolates and erucic acid (see Chapter 4). The
3.10d). In general, they contain about one glucosinolates (Figure 3.11a) are glycosides of
rhamnose for each 10 galacturonic acid units. I3-D-thioglucose with aglycones which yield
Galacturonic acid chains may be linked to both toxic isothiocyanate, thiocyanates, nitriles or
2 and 4 positions of the rhamnose unit and this oxazolidone derivatives such as goitrin under
results in a zig-zag chain. The pectin in prima- the influence of the enzyme glucosinolase
ry and secondary walls is called protopectin (myrosinase) which is released when the plant
and has more COOH groups esterified than in is crushed. Swelling of the thyroid (goitre) is a
the middle lamella. In the middle lamella the common symptom of glucosinolate poisoning.
COO· groups of the polygalacturonan are held Modern varieties have much lower levels of
together by Ca 2 + cross links. The degree of glucosinolates than earlier ones and the meal
cross linking and hence the strength of obtained from these varieties is therefore of
cell-cell adhesion may be regulated by the higher value.
Polysaccharides 33
a. /S-Glucose
CI-I =CH-CH-CH -C~
'"2 2 ~N-O-SO H
3
b. R1
I
Sugar -C- CN
I
R2
c.
oII
H3 C-C-NH Jr---- 0 COOH
OH
Figure 3.11 Sugars conjugated with other types of compound are quite commonly found in agricultural
materials. (a) Glucosinolates are found in some brassicas (e.g. rape) and may limit their usefulness as
crops. (b) The cyanogenic glucosides can be hydrolysed to yield toxic quantities of cyanide. (c) Sialic acid
is found in some cell membrane carbohydrates.
Cyanogenic glucosides Normally the glucosides are found in the

vacuoles of plants whilst the enzymes which
These are glycosides of a sugar and a cyanide- degrade them are located in the cytoplasm, so
containing aglycone. The most important negligible breakdown occurs. However, wilt-
cyanogens are amygdalin and prunasin (wild ing, frost or mechanical treatments bring the
cherries, almond, apricot, peach, apple ker- two together and cause degradation. The
nels), dhurrin (sorghum) and linamarin (white enzymes are also produced by rumen bacteria,
clover, cassava, linseed and lima beans) which so ruminants are particularly sensitive to these
have the general structure shown in Figure compounds.
3.11b. Exposure to these compounds affects live-
Cyanogens can be broken down by the stock which browse on the leaves of plants
action of glucosidases and hydroxynitrile lyas- such as chokecherry (Prunus virginiana) and
es to release HCN, which is extremely toxic Saskatoon serviceberry (Amelanchiar alnifolia).
because it inhibits cytochrome oxidase which Some varieties of cassava also contain
catalyses the final step in the electron trans- cyanogens which are poisonous to humans
port chain (see Chapter 12). who eat it. Traditional methods of preparation
minimize the risk of poisoning and involve contain similar proteins which may serve
either grating and soaking to remove the similar functions.
HeN, or cooking to destroy the enzymes and Many mammalian cell membranes have pro-
prevent cyanide production. jecting carbohydrate groups on their surfaces,
and it is through these carbohydrates that
many of the processes of cell recognition take
Lectins and their action
place. For instance, red cells from individuals
Lectins are proteins which are able to recog- with A-, B- or O-type blood groups differ in the
nise and bind to specific sugar sequences. nature of the carbohydrate groups exposed at
They may therefore agglutinate cells which the cell surface. The cells of the intestinal wall
bear carbohydrates on the outside of their also bear carbohydrate side chains, many of
walls, or precipitate glycoproteins (proteins them mannose polysaccharides (mannans). On
that contain one or more carbohydrate their outer cell membranes, the infecting bacte-
groups bound to them). Many lectins of ria carry lectins which are able to recognise and
plant origin are known. Thus concanavalin A bind to the specific sequences of carbohydrates
from jack bean (Canavalia ensiformis) recog- in the tissues that they are about to invade. The
nises oligomannosyl N-linked sugars, wheat- extent of bacterial invasion can be reduced in
germ agglutinin binds sialic acid (Figure animals fed high levels of dietary mannans.
3.11c) and N-acetylglucosamine, and ricin This has the effect of occupying all of the lectin-
from castor bean bind galactose. These com- binding sites so that they are unable to lock
pounds are of interest to science as probes of onto the intestinal polysaccharides.
carbohydrate structure and function, espe- Promising results have been obtained in
cially in relation to membrane structure, but feeding complex polysaccharides of glucose
at a more practical level many of them are and mannose (glucomannans) extracted from
toxic constituents of feeds. Within the organ- the cell walls of cultivated yeasts. Reductions
ism they have important functions in cellular in the incidence of infectious respiratory and
recognition. One example of this is in devel- enteric illness have been demonstrated in
opment of self-incompatibility in plants: ger- poultry, pigs and pre-ruminant calves. The
mination of pollen grains is prevented in advantages for animal health are expected to
many types of plants if lectins in the pollen be confined to non-ruminant animals which
recognise the stigma as being from the same do not have the capability to break down glu-
plant. It is also now recognised that animals comannans in the gut.
FATTY ACIDS AND LIPIDS 4
4.1 Introduction 35
4.2 Structure and occurrence of lipids 35
4.2.1 Fatty acids 35
4.2.2 Triacylglycerols and other acylglycerols 39
4.2.3 Glycerophospholipids 42
4.2.4 Glycosylglycerides 43
4.2.5 Sphingolipids 44
4.2.6 Terpenes and steroids 45
4.2.7 Waxes 47
4.1 INTRODUCTION erties are determined by the type of fatty acids

The term lipids is used to describe a chemical- incorporated into the triacylglycerols, as
described in more detail later in this chapter.
ly heterogeneous group of organic com-
pounds which have in common the general
property that they are insoluble in water but 4.2 STRUCTURE AND OCCURRENCE OF
are soluble in organic solvents such as hydro- LIPIDS
carbons (e.g. hexane and toluene), chloroform
4.2.1 FATTY ACIDS
and alcohols. In its widest sense the term
encompasses natural products such as the fat- Fatty acids are a group of aliphatic carboxylic
soluble vitamins, carotenoids, steroids, ter- acids which can contain from two to 24 or
penes, bile salts, fatty acids and their ester and more carbon atoms. The most abundant types
amine derivatives. Commonly it is used more of fatty acids are saturated and unsaturated
narrowly to include only fatty acids and their straight-chain fatty acids. Other types of fatty
derivatives, waxes, steroids and steroid esters. acids, such as branched-chain fatty acids,
Several other terms in common use need to hydroxyl-substituted fatty acids and cyclic
be defined more precisely for the purposes of fatty acids, are usually minor components of
this book. 'Fat' is often used in a very general lipids.
sense to mean any substance that is fatty in
texture. Similarly, in common usage 'oil' is a
term used to describe liquids as different as Saturated and unsaturated straight-chain
cooking oil and engine oil. In the context of fatty acids
this chapter, and for most nutritional and bio- Saturated and unsaturated fatty acids usually
chemical applications, fats and oils refer to tri- contain an even number of carbon atoms, typ-
acylglycerols (triglycerides in older texts) in ically between 10 and 24 carbon atoms in most
which a fatty acid is esterified to each of the plant and animal tissues. Small amounts of
hydroxyl groups of the trihydric alcohol glyc- odd-numbered fatty acids (mainly 15 and 17
erol. It is generally accepted that, at room tem- carbon atoms) are also found in plant and ani-
perature, fats are solids such as lard or drip- mal lipids. Unsaturated fatty acids contain
ping whereas oils are liquids such as rapeseed one or more double bonds. Fatty acids with
oil or olive oil. These different physical prop- one double bond are called monounsaturated
36 Fatty acids and lipids
fatty acids, those with more than one double described in Chapter 19. When using the .:l
bond are referred to as polyunsaturated fatty notation it is usual to indicate the configura-
acids or PUFAs. The double bonds may have tion of the double bonds in the full systematic
one of two configurations, cis or trans (Figure name. Thus palmitic acid, a saturated fatty
4.1). As a general rule the double bonds in acid with 16 carbon atoms, is the trivial name
most naturally occurring unsaturated fatty for hexadecanoic acid which is also referred to
acids have the cis configuration, although as C16:0 using a shorthand notation. Linoleic
fatty acids with trans double bonds are found acid is the trivial name for a fatty acid which
in bacterial lipids. PUFAs with a combination contains 18 carbon atoms and two cis double
of both cis and trans double bonds are pro- bonds, one between carbons 9 and 10 and the
duced from cis unsaturated fatty acids by other between carbons 12 and 13. Its systemat-
chemical hydrogenation of vegetable oils, ic name is all-cis .:l9,12 octadecadienoic acid
during the manufacture of margarines and which in this book is represented in the short-
during biohydrogenation of dietary PUFAs in hand notation as .:l9,12 C18:2 or CI8:2n-6.
the rumen of ruminant animals. Prior to the introduction of the numbering
Fatty acids are named in a number of dif- system, the Greek alphabet was used to label
ferent ways. For the most commonly occurring fatty acid carbon atoms. The lettering system
fatty acids trivial names are often used, did not label the carboxyl carbon, thus, the a-
although a systematic naming convention can carbon was equivalent to carbon 2. The methyl
be used for all fatty acids indicating the num- carbon was always referred to as the w-carbon.
ber of carbon atoms and the number, type and Although this lettering system is no longer in
position of double bonds and substituent use in modern texts it is closely linked with the
groups. Carbon atoms in a fatty acid are nor- discovery of the pathways for the metabolism
mally identified with respect to the carboxyl of fatty acids, and it is for this reason that the
carbon, which is carbon 1. The position of the three pathways of fatty acid oxidation are still
double bonds is identified by one of two nam- referred to as a-, 13- and w-oxidation (see
ing conventions. In the first convention, dou- Chapter 13).
ble bonds are numbered from the carboxyl car- Table 4.1 contains a list of saturated and
bon, and their position indicated by the nota- unsaturated fatty acids commonly found in
tion .:lx (where x is the number of carbon atoms plant and animal tissues, with their common
between carbon 1 and the double bond). The name, systematic name, shorthand notation
second convention uses the number of carbon and chemical structure.
atoms between the methyl carbon and the The presence of a cis double bond markedly
nearest double bond and uses the n- (n minus) reduces the melting point of a fatty acid, as can
notation. This convention is particularly useful be seen from Table 4.2. This depression occurs
for identifying families of fatty acids derived because the cis double bond introduces a bend
from a common precursor fatty acid, as into the otherwise linear structure of a saturat-
ed fatty acid, preventing the molecules from
H H H stacking closely together into a crystalline
I -C-C-
I I structure as the temperature decreases. The
greater the number of double bonds in a fatty
-C===C-
I
H
acid of a given chain length, the lower its melt-
ing point. In contrast, a trans double bond does
not change the linear nature of a fatty acid and
trans cis has a much smaller effect on melting point.
Figure 4.1 The configuration of trans and cis dou- Lauric and myristic acids are minor compo-
ble bonds. nents of most animal fats, but occur in signifi-
Table 4.1 Nomenclature and structure of commonly occurring fatty acids
Trivial name Systematic name Shorthand notation Structure

Saturated fatty acids
Lauric acid Dodecanoic acid C12:0 CH,(CH')lOCOOH
Myristic acid Tetradecanoic acid C14:0 CH3(CH')12COOH
Palmitic acid Hexadecanoic acid C16:0 CH,(CH2)14COOH
Stearic acid Octadecanoic acid C18:0 CH3(CH')16COOH
Arachidic acid Eicosanoic acid C20:0 CH3(CH2)18COOH
Behenic acid Docosanoic acid C22:0 CH,(CH')2IlCOOH
Lignoceric acid Tetracosanoic acid C24:0 CH,(CH2lzzCOOH
Monounsaturated fatty acids

Palmitoleic acid cis-a9-hexadecenoic acid Ll.9CI6:1 CH 3(CH2),CH=CH(CH2),COOH
Oleic acid cis-a9-octadecenoic acid Ll.9C18:1 CH 3(CH 2),CH=CH(CH2),COOH
Gondoic acid cis-a l1-eicosenoic acid Ll.11C20:1 CH 3(CH2),CH= CH(CH2)9COOH
Erucic acid cis-a13-docosanoic acid Ll.13C22:1 CH3(CH2),CH=CH(CH2)IICOOH
Nervonic acid cis-a15-tetracosenoic acid 1I.15C24:1 ' CH3(CH2),CH=CH(CH2)13COOH
Polyunsaturated fatty acids

all cis-Ll.6,9-octadecadienoic acid Ll.6,9CI8:2 CH 3(CH,),CH=CHCH,CH=CH(CH 2),COOH
Linoleic acid all cis-Ll.9,12-octadecadienoic acid 1I.9,12CI8:2 CH 3(CH2),CH=CHCH 2CH=CH(CH2),COOH
'V-Linolenic acid all cis-Ll.6,9.1 2-octadecatrienoic acid Ll.6,9.1' C18:3 CH,(CH2),CH=CHCH 2CH=CHCH 2CH=CH(CH 2),COOH
a-Linolenic acid all cis-Ll.9,12,I'-octadecatrienoic acid Ll.9,12,I' C18:3 CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2),COOH
Arachidonic acid all cis-Ll.',8,11,I'-eicosatetrenoic acid 11.',8,11,1' C20:4 CH,(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3COOH
EPA all cis-Ll.,,8.1U 4,I'-eicosapentaenoic acid 1I.,~,l1.1"I' C20:5 CH3CH,CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH,)3COOH
all cis-Ll.,,10,13,16.1 9-docosapentaenoic acid 11.7.10,13,16,19 C22:5 CH,CH,CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2),COOH
DHA all cis-Ll.,,',10,13,16,19-docosahexaenoic acid 6.4,7,10,13,16,19 C22:6 CH 3CH,CH=CHCH2CH=CHCH2CH=CHCH,CH=CHCH,CH=CHCH2CH=CH(CH')2COOH
Table 4.2 Melting point of commonly occurring fatty acids
Trivial name Shorthand notation Melting point (OC) *
Palmitic acid C16:0 60.7

Palmitoleic acid cis-d9-C16:1 1.0
Stearic acid C18:0 69.6
Oleic acid cis-d 9-C18:1 16.0
Elaidic acid trans-d 9-C18:1 44.0
Linoleic acid all cis-d 9,12-C18:2 -5.0
a-Linolenic acid all cis-d9,12,15_C18:3 -11.0
Arachidic acid C20:0 75.4
Gondoic acid cis-d 1c C20:1 24.0
Erucic acid cis-d 13-C20: 1 24.0
Arachidonic acid all cis-d5,8,1l,14-C20:4 -49.5
*From Gurr, M.l. and Harwood, J.L. (1991) Lipid Biochemistry, An

Introduction, 4th edn, Chapman & Hall, London.
cant quantities in certain vegetable oils such as Although many monounsaturated fatty
coconut and palm-kernel oils. The properties acids have been identified, by far the most
of these oils make them useful ingredients in abundant is oleic acid, which is found in vary-
the food industry. Coconut oil is used exten- ing proportions in all animal and plant lipids. It
sively as an ingredient of calf and lamb milk is the major fatty acid of olive and almond oils.
replacer due to its high digestibility. When Other monoenoic fatty acids are gondoic acid
included in ruminant feeds the lauric acid oils and erucic acid (d l l C20:1 and dl3 C22:1) which
can adversely affect rumen function. The are found in oils from seeds of the genus
shorter-chain fatty acids (C4:0; C6:0; C8:0 and Cruciferae. In certain varieties of rape and mus-
CI0:0) are characteristic of milk fats. Some tard, erucic acid may constitute in excess of 50%
species of mammals produce milk which is of the fatty acids. Because of the toxic properties
particularly rich in these acids. For example, in of this fatty acid, varieties of rape grown for
fat from rabbit milk, C6:0 and C8:0 constitute human and animal consumption have been
more than 40% of the total fatty acids, where- bred that are low in erucic acid ( < 5.0%).
as C8:0 and CI0:0 make up more than 60% of PUFAs are found in both plant and animal
the fatty acids of elephant milk. lipids. The C18 PUFAs, linoleic and a-linolenic
Palmitic acid is the major saturated fatty acids, are found in many vegetable oils. Rich
acid found in most vegetable oils and is also sources of linoleic acid are soyabean oil, maize
found in significant quantities (15-25% ) in ani- (corn) oil and safflower oil which contain typ-
mal fats. Stearic acid is the predominant satu- ically 50-75%. These oils also contain lower
rated fatty acid in ruminant fats, accounting proportions of a-linolenic acid. The best-
for as much as 40-45% of the total fatty acids. known plant source of a-linolenic acid is lin-
In pig and poultry fats it occurs in lower pro- seed (flax) oil. 'Y-Linolenic acid is usually a
portions and it is often only a minor compo- trace component of plant and animal fatty
nent in fish and vegetable oils. Longer-chain acids, but it is found in significant quantities in
saturated fatty acids (C20:0, C22:0 and C24:0) the seeds of the evening primrose, borage and
occur as only trace components in both animal blackcurrant. Although its medicinal proper-
and vegetable lipids ties are not understood, consumption of oils
Structure and occurrence of lipids 39
rich in this fatty acid appears to have beneficial Trace quantities of iso and anteiso fatty acids
effects on sufferers of multiple sclerosis. In (0.1-03%) are found in ruminant animal fat
general, plant lipids do not contain significant depots. They arise as a result of the digestion
amounts of PUFAs with more than 18 carbons. and absorption of the lipids from rumen
However, examination of animal tissue lipids microorganisms as they pass through the
reveals a more complex picture, with a range small intestine. When certain unusual diets are
of PUFAs with chain lengths from 18 to 22 car- provided the proportion of branched-chain
bon atoms. Fatty acids in fat depots (adipose fatty acids in sheep and goat fat can be greatly
tissue) have a relatively simple fatty acid com- increased (see Chapter 19).
position containing ,;l9,12 C18:2 and some ,;l9,12,15
C18:3 as the main classes of PUFAs, whereas Hydroxy and cyclic fatty acids
other tissues such as liver and muscle tissue
contain a wider range including ,;l9,12 CI8:2; A number of hydroxy fatty acids are found in
,;l9,12,15 CI8:3; ,;l5,8,1l,14 C20:4; ,;l5,8,1l,14,17 C20:5 and
bacterial lipids. These are mainly saturated in
,;l4,7,lO,13,16,19 C22:6, plus many other minor com-
nature, such as 3-hydroxymyristic acid, and are
ponents. This complexity reflects the higher found predominantly in the lipopolysaccharide
fraction of the cell membrane. The best known
proportion of membrane phospholipids found
in these tissues, Most marine oils contain sub- example of a hydroxy fatty acid in plant lipids
stantial amounts of the longer-chain C20 and is the occurrence of ricinoleic acid (12-hydroxy-
C22 PUFAs, particularly; ,;l5,8,1l,14,17 C20:5, oleic acid) which constitutes between 80 and
,;l7,10,13,16,19 C22:5 and ,;l4,7,lO,13,16,19 C22:6. Typical
95% of the fatty acids in castor oil.
fatty acid compositions of vegetable and ani- Cyclopropane and cyclopropene fatty acids
mal lipids are given in Table 4.3. are found in a number of plant species, particu-
Although saturated and unsaturated larly the Malvaceae and Sterculaceae, e.g, mal-
straight-chain fatty acids make up by far the valic acid and sterculic acid which is a minor
greatest proportion of those found in plant component of cottonseed oil (Figure 4.3),
and animal tissue lipids, there are a number of
other important, minor types which can have 4,2.2 TRIACYLGL YCEROLS AND OTHER
an impact on the properties of lipids and, in ACYLGL YCEROLS
some cases, their nutritional value,
Most purified fats and oils isolated from plant
and animal sources and used in human and
Branched-chain fatty acids
animal diets are triacylglycerols. The term tria-
This term is normally used to describe fatty cylglycerol encompasses a wide spectrum of
acids which contain one or more methyl (and molecular species in which each of the three
rarely ethyl) substituents along the carbon hydroxyl groups of glycerol are esterified to a
chain. Many microorganisms contain fatty acid (Figure 4.4).
branched-chain fatty acids which are mainly Because of the wide variety of fatty acids
of the iso and anteiso type (Figure 4.2). that occur naturally, many thousands of mole-
These fatty acids are typical of most Gram- cular species of triacylglycerols are possible.
positive and some Gram-negative organisms, Those which contain three identical fatty acids
Iso-branched fatty acids Anteiso-branched fatty acids
Figure 4.2 The structure of iso- and ante iso-branched chain fatty acids,
Table 4.3 Fatty acid composition of vegetable and animal lipids
Lipid ClO:0 C12:0 C14:0 C16:0 C16;1~ C18:0 C18:1~ C18:2·~ C18:3I C20,0 C20:1§ C20:4§ C20:5~ C22:1§ C22:5§ C22,@
Vegetable lipids
Coconut oil* 7.0 47.0 17.0 8.0 4.0 5.0 2.0
Palm kernel oil * 4.0 51.0 17.0 8.0 2.0 13.0 2.0
Palm oil* 1.0 48.0 4.0 38.0 9.0
Cottonseed oil * 1.1 27.3 1.4 3.1 16.7 50.4
Maize oil* 12.6 0.8 1.8 30.0 54.3 0.5
Olive oil* 14.0 2.0 2.0 64.0 16.0
Rape oil (low erucic 4.0 2.0 56.0 26.0 10.0 2.0
acid)t
Rape oil (high erucic 3.0 1.0 16.0 14.0 10.0 1.0 6.0 49.0
acid)t
Safflower oil * 6.6 0.6 3.4 12.2 77.0 0.2
Soyabean oil * 12.0 3.6 23.7 51.4 8.8
Sunflower oil * 6.0 3.0 27.0 64.0
Linseed oilt 6.1 0.1 3.2 16.1 14.2 59.8
Animal lipids
Beef tallow* 3.0 26.0 6.0 17.0 43.0 4.0
Lard* 2.0 26.0 4.0 14.0 43.0 10.0
Chicken fatb 1.2 20.3 3.9 6.5 33.5 18.5 2.6 0.2 0.5 0.3 0.4
Sheep liver 0.8 17.0 1.1 29.3 15.4 9.5 1.8 6.9 1.4 2.6 5.2 1.9
Chicken liverb 0.8 22.9 1.9 14.5 25.5 8.8 0.5 0.4 2.4 1.8 0.3 2.0
Chicken breast muscleb 1.4 19.9 3.6 9.0 25.5 17.5 1.6 0.4 2.8 0.7 1.1 2.1
Cod liver oil * 4.0 10.0 12.0 20.0 15.0 12.0 11.0
Menhaden oil * 6.0 18.0 12.0 5.0 16.0 4.0 4.0 13.0 13.0
* From Allen, J.e. and Hamilton, R.J. (eds) (1989) Rancidity in Foods, 2nd edn, Elsevier Applied Science, London and New York.
t From Gunstone, FD., Harwood, J.L. and Padley, F.B. (1994) The Lipid Handbook, 2nd edn, Chapman & Hall, London.
b From Scaife, J.R., Moyo, J., Galbraith, H.,Michie, W. and Campbell, V. (1994) Effect of different dietary supplemental fats and oils on the growth performance
and tissue fatty acid composition of female broilers. British Poultry Science, 33, 107-118.
§ C16:1 = ~7 C16:0; C18:1 = ~9 C18:1; C18:2 = ~9,12 C18:2; C18:3 = ~9,12,15 C18:3; C20:1 = ~11 C20l; C20:4 = ~5,8,11,14 C20:4, C20:5 = ~5,8,11,14,17 C20:5;
C22:5 = ~7,10,13,16,19 C22:5 and C22:6 = ~4,7,10,13,16,19 C22:6
they contain. Fats such as beef and pork tallow
(lard) contain a higher proportion of saturated
fatty acids than plant oils. The degree of unsat-
uration of fats and oils is often measured as the
iodine value (IV), a measure of the reaction of
Sterculic acid iodine with the double bonds of unsaturated
Figure 4.3 The structure of sterculic acid. fatty acids. High iodine values indicate a high
degree of unsaturation. The saponification
value (SV) of triacylglycerols gives compara-
constitute a relatively small proportion of the tive information about the chain length of the
naturally occurring triacylglycerols; the vast fatty acids they contain. It represents the yield
majority contain at least two different fatty of fatty acid from one gram of triacylglycerol.
acids and are called mixed triacylglycerols. High values indicate the presence of signifi-
The great economic importance of triacyl- cant amounts of short- and medium-chain
glycerols for industrial, pharmaceutical and length fatty acids (see Table 4.4).
food use can be seen in Table 4.4. Saponification is the process in which lipids
The physical properties of triacylglycerols are hydrolysed by heating in dilute ethanolic
are determined by the nature of the fatty acids KOH. The fatty acids released form water-sol-
Glycerol
Triacylglycerol backbone
where R1 • R2 and R3 are usually
long chain saturated and unsaturated
fatty acids
Shorthand notation
for triacylglycerols
Figure 4.4 The outline structure of triacylglycerols (where R1, Rz and R3 are usually long-chain saturated
and unsaturated fatty acids), showing the glycerol backbone and the shorthand notation used to repre-
sent triacylglycerols.
Table 4.4 World production and physicochemical properties of fats and oils
Fat/oil Annual Melting S/U Saponification Iodine value

production point ratio· value
(tonnes)#
Beef tallow } 40-50 0.85 190-200 32-47

Pig tallow }1O.6 x 106 28--48 0.72 193-200 46-66
Butter 6.3 x 106 28-33 1.70 216-235 26-45
Cod liver oil } 0.16 182-193 155-170
Menhaden oil } 1.4 x 106 0.41 189-193 160-180
Coconut oil 3.4 x 106 23-26 13.29 251-264 7-10
Palm kernel oil 1.9 x 106 25-30 8.09 244-254 14-20
Palm oil 11.0 x 106 38--45 1.13 196-202 48-56
Cottonseed oil 3.3 x 106 0.46 191-196 100-112
Maize oil 0.4 x 106 0.17 187-196 84-102
Olive oil 1.9 x 106 0.19 187-196 117-130
Rapeseed oil'!' 7.8 x 106 173-181 105-120
Soyabean oil 16.5 x 106 0.18 189-195 124-133
Sunflower oil 8.0 x 106 0.09 186-194 127-136
Linseed oil 0.8 x 106 0.11 188-195 180-185
# 1990 figures from Agra Europe (1993) No 1537. Agra Europe (London) Ltd, Tunbridge Wells.
* Ratio of saturated to unsaturated fatty acids.
'" Low erucic acid rapeseed oil.
uble potassium salts (soaps), hence the term Other acylglycerols, such as diacylglycerols
saponification. and monoacylglycerols, usually occur as
The distribution of fatty acids between the minor components of tissue lipids and are
three hydroxyl groups of glycerol is by no important intermediates in both the synthesis
means random. During the biosynthesis and and breakdown of triacylglycerols.
later modification of these molecules (dis- Diacylglycerols contain only two fatty acids
cussed in more detail in Chapter 19), the type and can exist in two forms, 1,2-diacylglycerols
of fatty acid esterified to each position (satu- and 1,3-diacylglycerols. The first species is the
rated or unsaturated, long-chain or short- initial breakdown product of triacylglycerols
chain) is determined by the specificity of the during lipid digestion in the monogastric ani-
enzymes which catalyse this addition. Thus, in mal. Monoacylglycerols, which contain only
general, fatty acids found in position 1 are pre- one fatty acid, can also exist in two forms, 1- or
dominantly saturated and those found in posi- 2-monoacylglycerols. In practice 2-monoacyl-
tion 2 are unsaturated. The fatty acids in posi- glycerol occurs most commonly. It is a major
tion 3 appear to have a more variable nature, end product of monogastric lipid digestion
although in mammals they tend to be rich in and the substrate for triacylglycerol resynthe-
PUFAs (e.g. il5,8,1l,14,17 C20:5, il4,7,IO,13,16,19 C22:6). sis in the intestinal mucosa (see Chapter 19),
In fish oils these fatty acids tend to occupy the
2 position. Milk fats which are synthesized in
4.2.3 GL YCEROPHOSPHOLIPIDS
the mammary tissue are characterized by the
presence of short- and medium-chain-Iength Glycerophospholipids are found in all living
fatty acids. These fatty acids are found mainly organisms. They are based on the structure of
in position 3. glycerol and are important components of bio-
erophospholipid, phosphatidic acid, the phos-
phate group is not linked to any other sub-
stituent group. Phosphatidic acid is normally a
minor constituent of tissue phospholipids
o
"
although it is an important intermediate in
O-p-o-x glycerophospholipid synthesis.
I
0- Most phospholipids contain a substituent
group linked to the phosphate in position 3.
These groups, a number of which are basic
compounds, are usually polar in nature, the
most common being choline, ethanolamine,
serine and inositol. The choline-containing
glycerophospholipid has traditionally been
called lecithin, and although this name is still
in use it is now more commonly referred to as
HQ-CH 2-CH-NH 2 phosphatidylcholine. Similarly those glyc-
I erophospholipids containing ethanolamine,
(c) eOOH serine and inositol are known as phos-
phatidylethanolamine (old name cephalin),
OH OH
phosphatidylserine and phosphatidylinositol,
~ ~~
respectively.
Because these molecules contain both
hydrophobic long-chain fatty acids and
H OH hydrophilic phosphate and substituent groups,
(d) OH H they have physicochemical properties which
Figure 4.5 The outline structure of glycerophos- make them ideal building blocks for mem-
pholipids. R] and R2 are usually long-chain saturat- branes. They are often described as amphiphilic
ed or unsaturated fatty acids. The X group may be molecules because of their ability to act as an
one of a number of compounds, the structure of interface between a polar aqueous environ-
some of which is shown: (a) choline ->
phosphatidylcholine; (b) ethanolamine -> ment and a non-polar lipid environment.
phosphatidylethanolamine; (c) serine -> In animals, in addition to fulfilling an impor-
phosphatidylserine; (d) inositol -> phosphatidyli- tant structural role, membrane glycerophos-
nositol. pholipids have a role in inter- and intracellular
signalling by acting as a reservoir of PUFAs,
logical membranes. Their structures can be which are precursors for the synthesis of a
represented as shown in Figure 4.5. family of related compounds such as
Hydroxyl groups in the 1 and 2 positions of prostaglandins, leukotrienes and thrombox-
the glycerol backbone are esterified to fatty anes. These compounds are powerful local reg-
acids, usually long-chain fatty acids. In bacte- ulators involved in processes as varied as the
ria and animals the distribution of fatty acids inflammatory response, platelet aggregation,
between the 1 and 2 position is similar to that smooth muscle contraction and ovulation.
of triacylglycerols, i.e. predominantly saturat-
ed fatty acids in position 1 and unsaturated
fatty acids in position 2. Plants, however, do
4.2.4 GL YCOSYLGL YCERIDES
not show the same consistent pattern of fatty
acid distribution. These glycerol-based lipids are characterized
The hydroxyl group in the 3 position is by the presence of sugar residues. They are
phosphorylated. In the simplest form of glyc- found in small quantities in bacteria and ani-
mals, but are most characteristic of photosyn- The glycosylglyceride fraction from chloro-
thetic tissues in plants, algae and cyanobacte- plasts is also characterized by the presence of
ria, particularly as components of chloroplast sulphoquinovosyldiacylglycerol, so-called
membranes (Figure 4.6). plant sulpholipid, which contains a sulphate
In plants the most commonly occurring group on the 6 carbon of the sugar residue. A
species are the mono- and digalactosyl diacyl- number of other sulpholipids are found as
glycerols. These contain either a single galac- minor constituents of algae and bacteria.
tose residue or two galactose residues (linked Glycosylglycerides have a high content of
together by an a-1,6 glycosidic bond) esterified polyunsaturated fatty acids, particularly a9,12
to the 3 position of the glycerol backbone. In C18:2 and a9,12,15 C18:3 and in some cases, such
plants galactose is almost exclusively the sugar as spinach chloroplast monogalactosyldiacyl-
found in glycosylglycerides, but in algae and glycerol, the unusual fatty acid a7,10,13 C16:3.
bacteria diglycosyldiacylglycerols are found These lipids form the major source of dietary
which contain other sugars, mainly two glu- fatty acids in grazing animals.
cose or two mannose residues which may be
linked a-1,2 or 13-1,6.
4.2.5 SPHINGOLIPIDS
Sphingolipids are structural lipids found
mainly in membranes. They are based on the
structure of the long-chain amino alcohol
o 0 OH
sphingosine (Figure 4.7).
HO
OH
CH.(CH2)I2CH-CHCH-CHCH20H
(a) OH I
(a) NH2
OH
CH.(CH')'2CHzCHCH-CHCH20H
I
NH
I
c=o
I
(b) R
.
OH
CH.(CH2)'2CHaCHCH'9HCH2-0-(Sugar)n
NH
I
c=o
I
(e) R
9H ~ •
CH.(CH2)'2CH=CHCH'9HCH20-~:'-CH;CH2N (CH.l.
NH 0
I
c=o
o
I
0 OH (d) R
HO Figure 4.7 The outline structure of sphingolipids.

(a) Sphingosine. (b) Ceramide (N-acyl-sphingenin
- R is a long-chain saturated or unsaturated fatty
(c) OH acid. (c) Cerebrosides and gangliosides - (cerebro-
Figure 4.6 The outline structure of (a) monogalac- sides may contain a number of sugar residues,
tosyldiacylglycerol, (b) digalactosyldiacylglycerol gangliosides are characterized by the presence on
and (c) sulphoquinovosyldiacylglycerol (plant one or more residues of sialic acid (N-acetylneu-
sulpholipid). raminic acid). (d) Sphingomyelin.
The attachment of a fatty acid to the amino um oil, lemon oil, mint oil, turpentine, cam-
group of sphingosine produces N-acylsphin- phor oil and caraway oil, respectively.
gosine, or ceramide. Various other substituent An example of a sesquiterpene commonly
groups can be esterified via the primary alco- found in essential oils is farnesol. Perhaps one
hol group. In sphingomyelin the substituent of the most commonly occurring diterpenes is
group is phospho choline and this lipid may, the phytol component of chlorophyll.
therefore, also be classified as a phospholipid. Squalene is probably the most abundant linear
Most other types of sphingolipids contain one triterpene (Figure 4.9).
or more sugar residues linked to N-acylsphin- Other higher terpenes in plants include the
gosine. The most commonly occurring sugars carotenoids such as (3-carotene (Figure 4.1Of),
are glucose, galactose and N-acetylglu- and the xanthophylls such as lutein. These
cosamine, usually linked by (3-1,4 or (3-1,3 gly- compounds are strongly coloured and are
cosidic bonds to form mono-, di-, tri- and often used as colorants in foods. For example,
tetraglycosylceramides. These sphingolipids the synthetic xanthophyll, carophyll yellow, is
are classified under the generic name cerebro- added to poultry diets to intensify the yellow
sides. Gangliosides are a further type of sphin- colour of egg yolk, and astaxanthin is added to
golipid similar to cerebrosides but containing commercial salmon diets to reproduce the
one or more residues of sialic acid (N-acetyl- pink flesh found in wild salmon. Flowers are a
neuraminic acid) normally linked (3-2,3 to a rich source of pigments and marigolds are
galactose residue. The names of sphingolipids grown in large quantities for the carotenoids
reflect their association with nervous tissue and xanthophylls they contain.
and particularly brain lipids, however they are Among the most important terpenes in the
widely distributed in other animal tissues and animal kingdom are three fat-soluble vitamins,
are found in small quantities in plants, A, E and K (Figure 4.10). In addition, the other
although gangliosides appear to be unique to fat-soluble vitamin, vitamin D, is related to the
the animal kingdom. terpenes as it is synthesized from sterols.
Another important group of terpenoid
compounds function as coenzymes in a num-
4.2.6 TERPENES AND STEROIDS
ber of oxidation-reduction reactions in nearly
Terpenes and sterols are related compounds all living organisms. This is the ubiquinone
which are constructed from the five-carbon coenzyme Q (CoQ), family of compounds. In
building block isoprene (Figure 4.8). higher organisms these are located mainly in
Terpenes containing two isoprene units are the mitochondria and are components of the
called monoterpenes, those containing three electron transport chain (see Chapter 12).
isoprene units are called sesquiterpenes, and These compounds contain a substituted
those containing four, six and eight isoprene quinone ring which can be reduced and reoxi-
units are called diterpenes, triterpenes, and dized, and a long isoprenoid side chain. The
tetraterpenes, respectively. Terpenes may be length of the side chain differs from organism
linear or cyclic molecules and some terpenes to organism. Similar compounds called plasto-
contain structures of both types. quinones occur in plant chloroplasts and are
A large number of terpenes have been dis- involved in photosynthesis (Figure 4.10).
covered in plants and many of these com- Yet another important class of terpenes is the
pounds have characteristic smells or flavours, polyprenols. These compounds are long-chain
and are major components of the essential oils linear polyisoprenes with a terminal alcohol
derived from such plants. The monoterpenes group. Certain polyprenols have an important
geraniol, limonene, menthol, pinene, camphor role to play in the synthesis of cell walls in bac-
and carvone are major components of gerani- teria and plants, and in the synthesis of cell-sur-
N
CH 3
I
~C, .?CH2
H2C CH
(a)
CH 3 CH 3
CH 3 I I
I
/C~ /CH2OH
C
HC/~CH
C
HC~
I I
H2C CH 'CH
I' C "
HaC"',
CH 3
1
H2C ,
I H2C, /CH 2
2
H2C , /CH2
CH CH
CH
C "
H C........... 'CH
3 3
I
C
H2c-:P 'CH 3
(b) (c) (d)
(e) (f) (9)

Figure 4.8 The structures of (a) isoprene, the basic five-carbon building block of terpenes, and some natu-
rally occurring monoterpenes in plant oils: (b) a-pinene, (c) geraniol, (d) limonene, (e) camphor, (f) men-
thol and (g) carvone.
face lipopolysaccharides, peptidoglycans and incorporation into macromolecules such as gly-

glycopolysaccharides. Compounds such as bac- coproteins.
toprenol are found mainly in bacteria and plant A number of important plant hormones are
tissues. The dolichols, found mainly in animal also terpenoids, for example abscisic acid and
plasma membranes, are involved in the trans- the gibberellins. These are discussed in more
port of glycopeptides across the cell membrane. detail in Chapter 27.
They are thought to act as carriers for the gly- A number of terpenoid compounds occur in
copeptides, transporting them to their site of insects. For example, compounds known as
Famesol
Phytol
Squalene
Figure 4.9 The structures of farnesol, phytol and squalene.
the juvenile hormones control the complicated portions, depending on the plant species and
post-embryonic development of insects. In tissue.
addition, a group of compounds known as the In animals the principal sterol is cholesterol.
ecdysones, found in insects and arthropods, In its free form and in the ester form it is a
control moulting. It is interesting to note that component of membranes and appears to play
ecdysones are also found in plants, and it has an important role in the modulation of mem-
been suggested that production of these com- brane fluidity. Cholesterol is the precursor for
pounds by plants is a defence mechanism the synthesis of a family of steroid hormones
which interferes with the development of found in animal tissues.
insect predators.
A variety of steroids are found in eukary-
4.2.7 WAXES
otic organisms but few are found in bacteria
and other prokaryotes. The basic building The strict chemical definition of a wax is an
block of steroids, which are derived from ester formed between a long-chain alcohol
squalene, is a fused, four-membered ring and a long-chain fatty acid. However, the term
structure, sometimes called the steroid nucle- is used more widely for plants and animals to
us. The four most commonly occurring describe the surface lipids of stems and leaves
steroids in plants and animals are shown in and the sebaceous secretions associated with
Figure 4.11. These compounds have a skin, hair and feathers. The main components
hydroxyl group on carbon number 3 and are of waxes are long-chain alkanes, acids, alco-
therefore steroid alcohols or sterols. hols and aldehydes and their esters.
Steroid esters can be formed by reaction of In plants these compounds form complex
a long-chain fatty acid with this hydroxyl polymers and constitute a significant propor-
group. Alternatively they can form glycosidic tion of suberin and cutin, the structural layers
links with sugars to give steroid glycosides. of the cuticle. It has been observed that the
In plants the most abundant sterol is sitos- alkane composition of plants can be used as a
terol. In addition, there are smaller amounts 'fingerprint' to identify plant species. Because
of stigmasterol, campesterol and cholesterol, these alkanes are not absorbed in the digestive
all of which occur as free sterols, steroid tract, analysis of alkanes in faeces can be used
esters and steroid glycosides in varying pro-
o
o
Vitamin ~ (Menaquinone) Ubiquinone
Plastoquinone
Vitamin A (Retinol)
Vitamin E (a-Tocopherol)
p-Carotene
Figure 4.10 The structures of some of the higher terpenes found in plants and animals: vitamin ~
(menaquinone); ubiquinone; plastoquinone; vitamin A (retinol); vitamin E (a-tocopherol); l3-carotene.
Structure and occurrence oj lipids 49
Cholesterol
Campesterol
HO
Sitosterol
HO
Stigmasterol
HO
Figure 4.11 The structures of the most common sterols found in plants and animals: cholesterol; campes-
terol; sitosterol; stigmasterol.
to establish the quantity and types of plants store, for example in some marine animals, in
eaten by grazing animals. zooplankton and in certain oil seeds, jojoba
In certain plant and animal species, waxes being the most often quoted.
may be used as an alternative form of energy
AMINO ACIDS AND PROTEINS 5
5.1 Introduction 51
5.2 Amino acids 52
5.2.1 Structure of amino acids 52
5.3 Non-protein amino acids and related 52
compounds
5.3.1 Canavanine 52
5.3.2 Selenium-containing amino acids 52
5.3.3 Mimosine 54
5.3.4 Lathyrogens 55
5.3.5 S-methyl cysteine sulphoxide (SMCO) 55
5.3.6 Alkaloids 55
5.4 Phenolics 57
5.4.1 Lignin 57
5.4.2 Tannins 57
5.4.3 Flavonoids 58
5.5 Peptide bonds 60
5.6 Protein function and structure 61
5.6.1 Primary protein structure 62
5.6.2 Secondary protein structure 62
5.6.3 Tertiary structure 63
5.6.4 Quaternary structure 64
5.7 Properties of proteins 66
5.7.1 Ionic strength and presence of specific 67
ions
5.7.2 Effect of pH 68
5.7.3 Denaturation 68
5.7.4 Effect of heat 69
5.8 Prions 69
5.1 INTRODUCTION genetic information carried in the DNA is able

to control the activities of the cell. Thus the syn-
Amino acids are small molecules containing thesis of proteins is a vital process in all cells,
both -NHz (amino) and -COOH (carboxylic and this requires adequate supplies of all of the
acid) groups. They are found in all types of amino acids of which they are composed.
cells. Most of the amino acid molecules are Although amino acids occur most common-
found linked together to form proteins. ly as components of proteins, some free amino
Proteins have vital functions including acting acids are also found in cells. The concentra-
as enzymes, as structural components of the tions of these are normally relatively low but
cell and in molecular recognition. It is through when plants are subjected to water or salt
the production of specific proteins that the stress, protein synthesis is slowed down and
52 Amino acids and proteins
some free amino acids, especially proline, may 5.3 NON-PROTEIN AMINO ACIDS AND
accumulate and reach quite high concentra- RELATED COMPOUNDS
tions.
In addition to the amino acids which are
found in proteins, there are also a large num-
5.2 AMINO ACIDS ber of other, non-protein amino acids which
exist in the free form, especially in plants.
5.2.1 STRUCTURE OF AMINO ACIDS
Several hundred non-protein amino acids
There are approximately 20 different amino have been extracted from plants and they are
acids which occur in proteins. They all have particularly widespread in legumes. Some of
the basic structure shown in Figure 5.la. them are toxic, or have physiological effects on
In all of the amino acids except glycine the animals and may be important components of
a-carbon atom is an asymmetric centre, so all animal feeds. Some examples are given in
amino acids except glycine are optically Figure 5.3.
active. All amino acids found in proteins are
the L-isomers. Although they are referred to
5.3.1 CANAVANINE
as amino acids, proline and hydroxyproline
do not contain a true amino group. Instead The non-protein amino acid canavanine is pre-
the nitrogen atom forms part of a five-mem- sent in jack bean (Canavalia ensiformis) seeds.
bered ring. This amino acid resembles arginine in struc-
In solution all free amino acids exist in the ture, and interferes with the metabolism of
form of zwitterions (doubly charged ions) as arginine and its incorporation into proteins in
shown in Figure 5.lb. animals which eat the seeds.
The amino acids can be divided into several
classes. Depending on the nature of the R
5.3.2 SELENIUM-CONTAINING AMINO ACIDS
group amino acids are classified as aliphatic,
hydroxy, sulphur-containing, aromatic, basic, Some plants growing on soils which are rich in
acidic and imino acids. The structures of the the element selenium may accumulate high
amino acids commonly found in proteins are levels of selenium-containing amino acids, in
shown in Figure 5.2. The side chain (R) can which selenium replaces the sulphur atom
also carry charges and its nature is important which normally forms part of their structure.
in determining whether the amino acid is Thus they may contain amino acids such as Se-
hydrophobic or hydrophilic, as well as deter- methylselenomethionine or Se-methylseleno-
mining the properties of any proteins in which cysteine (Figure 5.3). Such plants are toxic to
it is found. livestock eating them and may cause 'alkali
a-carbon
H/ atom
I
NHr C-C0 2H
I
R
Figure 5.1 The structure of an amino acid in (a) the un-ionized form and (b) the zwitterion form.
Aliphatic
Hydroxyl-containing Sulphur containing
H H H H H H H H H
NH3+1-c02- NH3+1-c02- NH3+1-c02- NH/1-e02- NH3+1-c0 2- NH 3+1-c0 2- NH3+1-c02- NH3+1-c02- NH3+--t-e02-
I
H tH3 tH-CH 3 tH2 tH-CH 3 tH 20H dH-OH tH 2SH tH2
I
t H3 tH-CH 3 tH2 CH 3 tH2
tH3 tH3 ~CH3
Serine Threonine Cysteine Methionine
Glycine Alanine Valine Leucine Isoleucine
Ser Thr Cys Met
Gly Ala Val Leu lie
S T C M
G A V L I
Acidic Imino acids

Aromatic
Basic H H
H H H CO2-
H H H NH 3+1-c02- NH 3+1-c02- +HN - t H
NH3+1-c0 2- NH3+1-c02- NH3+1-c02-
NH3+1-c02- NH 3+1-c0 2- NH3+1-c0 2- tH2 tH2
tH2 tH2 tH2
tH2 tH2 tH2 2- tH2
t0 I _
V
tH2 tH2 (CONH 2) CO 2
tH2 tH2
h
+HN ~NH (CONH 2) Proline
I Pro
OH tH2 NH Aspartic acid Glutamic acid P
0 O~ ~H3+ t=NH 2+
Asp Glu
D E
I (Asparagine) (Glutamine)
NH2
(Asn) (Gin)
Phenylalanine Tyrosine Tryptophan
Lysine Arginine Histidine (N) (Q)
Phe Tyr Trp
F Y W Lys Arg His
K R H
Figure 5.2 The structures of the amino acids commonly found in proteins.
Se-methylselenocysteine
Canavanine
l3-aminopropionitrile
Mimosine
o NH2 NH2
t I I
CH:r-S-CHrCH-COOH NC-CHrCH-COOH
S-methyl cysteine sulphoxide l3-cyanoalanine

(SMCOl
Figure 5.3 The structures of some non-protein amino acids: canavanine; Se-methylselenocysteine; mimo-
sine; l3-aminopropionitrile; S-methyl cysteine sulphoxide (SMCO); l3-cyanoalanine.
disease' or 'blind staggers'. These amino acids 5.3.3 MIMOSINE

are toxic to animals because they are incorpo-
rated into proteins in place of the normal sul- Mimosine is a toxic amino acid present in
phur-containing amino acids, but the proteins leaves and seeds of the tropical legume
which are produced are inactive. The plants Leucaena leucocephala (Figure 5.3). Although the
which accumulate the amino acids have plant is potentially of high nutritional quality,
adapted to avoid these effects. Low levels of its uses are limited by the presence of this
selenium-containing amino acids, particularly amino acid. Some of the toxic effects may be
seleno-methionine, may be used in animal due to mimosine, but others are caused by a
feeds as a selenium supplement for livestock. product of its breakdown in the rumen, dihy-
Sometimes this is achieved by persuading roxypyridine (DHP). Leucaena is not toxic to
yeasts to form proteins containing selenium ruminants from Hawaii because their rumen
amino acids and then incorporating the killed microorganisms further degrade DHP to non-
yeasts in commercial feedstuffs. toxic products. Introduction of Hawaiian
Non-protein amino acids and related compounds 55
rumen bacteria into Australian animals pro- which make them are uncertain, although
tects them against mimosine poisoning. they may confer some resistance to animal or
insect attack.
Some alkaloids have dramatic physiological
5.3.4 LATHYROGENS
effects on man and animals and many, includ-
Lathyrism is a disease which is caused by eating morphine and nicotine (Figure 5.4), have
ing seeds of the genus Lathyrus, including pharmaceutical and medicinal uses and may
Lathyrus sativus, the chickpea and Lathyrus be of great commercial and sociological impor-
odoratus, the annual sweet pea. The chickpea is tance. The presence of alkaloids renders some
mainly consumed by humans and causes neu- plants toxic to man and to livestock, as
rolathyrism, whilst sweet peas cause osteo- described below.
lathyrism in livestock, especially horses. The pyrrolizidine alkaloids (Figure 5.4)
Symptoms of osteolathyrism are skeletal occur in Senecio species, and are the cause of
deformity and rupture of the aorta. It is caused poisoning by S. jacobea (ragwort) and other
mainly by ~-aminopropionitrile (Figure 5.3) species. Although ragwort is not very palat-
which interferes with cross-linking of lysine able it may be consumed when there is no
residues in collagen. other forage or when it has been dried, as in
Neurolathyrism is caused by compounds hay. Pyrrolizidine alkaloids are toxic as a result
such as ~-N-oxalyl a,~-diaminopropionic acid of conversion in the liver into toxic metabolites
which is found in chickpea, and ~-cyanoala such as pyrroles. Liver function is seriously
nine in common vetch (Figure 5.3). These impaired, the liver is reduced in size and other
compounds attack nerve cells and lead to organs may also be affected. Cattle and horses
weakness and eventual paralysis of the legs. are susceptible to this type of poisoning but
The effects are most common in man and are sheep are relatively very resistant, possibly
rarely seen in animals. because their liver is less able to convert the
alkaloids to pyrroles. Some pyrrolizidine alka-
loids have also been shown to be carcinogenic
5.3.5 S-METHYL CYSTEINE SULPHOXIDE
to livestock.
(SMCO)
Indole alkaloids include the ergot alka-
Brassicas contain SMCO (Figure 5.3) as well as loids, produced by fungi, which grow on the
glucosinolates (see Chapter 3). SMCO appears seed of some grasses and cereals. These alka-
to be the cause of severe anaemia in ruminants. loids are based on lysergic acid (Figure 5.4)
In brassicas, garlic and onion this amino acid and may cause convulsions, numbness, gan-
makes up 4-6% of dry matter. The most likely grene of extremities and reproductive defects
cause of the toxicity is not SMCO itself but in humans and livestock. Ergot is the name
dimethyl disulphide, which is produced from commonly used for fungi of Claviceps species,
it in the rumen. This compound may react with and a condition resulting from consumption
reduced glutathione, preventing it from proof ergot-infected grain is known as ergotism.
tecting haemoglobin against oxidation. Rye is particularly susceptible to attack by C.
purpurea, and infected rye caused many epi-
demics of ergotism in Europe in the Middle
5.3.6 ALKALOIDS
Ages.
Alkaloids are nitrogen-containing, basic com- Quinolizidine alkaloids (Figure 5.4) occur in
pounds which are found in many plants. The lupins (Lupin us spp.) and laburnum.
nitrogen normally forms part of heterocyclic Cultivated lupins have been selected to have
rings. Several thousand alkaloids have been low levels of these compounds, but wild
identified but their functions in the plants species have caused great losses of sheep as a
nicotine
morphine
co
The quinolizidine nucleus
Retronecine - - basis of quinolizidine alkaloids
basis of
pyrrolizidine
alkaloids lysergic acid
H~OO~
~:~o
CH 3
o
HO HO H~O~
~
HO OH
solanidine
solanine
Figure 5.4 The structures of some alkaloids: morphine; nicotine; retronecine (basis of pyrrolizidine alka-
loids); lysergic acid; the quinolizidine nucleus (basis of quinolizidine alkaloids); solanine and solanidine.
Phenolics 57
result of respiratory paralysis, especially in the scar tissue after wounding or abscission of
USA. leaves.
Steroid alkaloids are present in plants of the
genus Solanum such as potatoes, tomatoes and
5.4.1 LIGNIN
nightshades, and are therefore known as
solanum alkaloids. The glycoalkaloid solanine Lignin is a component of the plant cell wall
(consisting of the aglycone solanidine and a but is found in only small quantities in prima-
side chain of sugars - Figure 5.4) is present in ry cell walls. Extensive lignification is restrict-
potatoes and can cause poisoning of both ed to tissues such as xylem and phloem and
humans and livestock. Concentrations of alka- takes place only after growth has stopped.
loids are highest in green sprouts and green Lignins are formed by polymerization of
tubers. Solanum alkaloids cause irritation of coniferyl alcohol, sinapyl alcohol and p-
the gastro-intestinal tract, neurological impair- hydroxycinnamyl alcohol through a free radi-
ment (they inhibit acetylcholinesterase) and cal mechanism which results in random for-
have been suggested to be teratogenic. mation of bonds and forms a very complex
Larkspurs (Delphinium spp.) have probably structure (Figure 5.6).
been responsible for greater losses of cattle in Whilst the gymnosperms (and pterido-
the USA than any other plant. Their toxicity is phytes) have lignin which contains almost
due to the presence of polycyclic diterpenoid exclusively coniferyl alcohol, hardwood trees
alkaloids which cause respiratory paralysis. and dicotyledonous and monocotyledonous
crops contain comparable quantities of
coniferyl and sinapyl alcohols (Table 5.1). In
5.4 PHENOLICS
addition, monocots contain appreciable quan-
Phenolics are compounds containing aromatic tities of p-hydroxyphenyl residues.
rings substituted with -OH groups. They are Lignin is extremely resistant to either chem-
not amino acids, but as they contain phenyl ical or enzymatic attack. Materials with a high
rings which are synthesized in the same way lignin content are therefore durable structural
as the phenyl rings of the aromatic amino materials and have low digestibility when
acids, it is convenient to discuss them here. they are part of the diet.
Animals are unable to synthesize aromatic
compounds but plants can and may contain a
5.4.2 TANNINS
wide range of them. The structure of some
typical phenolic compounds is shown in Tannins are polyphenolic materials which are
Figure 5.5. These are often found as compo- able to precipitate proteins from solution. The
nents of more complex molecules such as name is derived from the use of extracts con-
phytoalexins, coumarins, anthocycanins, tan- taining such compounds to tan leather, mak-
nins or lignins. ing it more resistant to microbial attack, heat
The production of a number of phenolic and abrasion. Tannins contain o-dihydroxy-
compounds appears to be related to disease phenol groups which allow them to form
resistance in plants. Thus protocatechuic acid hydrogen bonds and hydrophobic bonds with
is present in onions which are resistant to the proteins such as collagen in animal skins.
smudge fungus Colletotrichum circinans but Precipitation of proteins by plant extracts is
absent from those which are susceptible, and significant to animals. It reduces the digestion
chi orogenic acid may be oxidized to fungistat- of proteins in feeds, both by inhibiting diges-
ic quinones in resistant plants. Ferulic acid is tive enzymes and by precipitating (and there-
found in suberin which protects the under- fore making unavailable) protein in the feed.
ground parts of plants and which is formed in Precipitation of proteins in the mouth is
COOH COOH
COOH
I I I
CH CH
CH
II II II
0 0
CH CH
CH
(lOCH.
OH
OH
cinnamic acid p-coumaric acid ferulic acid
Figure 5.5 The structures of some phenolic compounds commonly found in plants: cinnamic acid; p-
coumaric acid; ferulic acid.
responsible for the astringent (bitter) taste of phenolics, such as gallic acid, condensed with
fruits such as persimmon in their unripe state. glucose (Figure 5.7).
This has a major effect on palatability of feeds
and provides protection against herbivores.
5.4.3 FLA VONOIDS
Inhibition of enzymes also confers resistance
to attack by insects, bacteria etc., rendering Flavonoids are IS-carbon compounds which
wood more durable. Tannins in forage are widely distributed in plants. Their struc-
legumes reduce the incidence of bloat by pre- ture is based on the flavonoid ring system
cipitating bloat-inducing proteins. (Figure 5.8).
The presence of tannins in crops such as The most important groups of flavonoids
sorghum reduces the palatability and are the anthocyanins and the flavones.
digestibility of the protein and reduces value,
especially for feeding of non-ruminants.
Anthocyanins
Sorghum tannins inhibit amylase and trypsin
in the digestive tract. High-tannin lines are These are coloured compounds that commonly
generally more resistant to birds and normally occur in flowers and in certain types of fruits,
have pigmented seed coats. stems, leaves and even roots. In flowers, they
Tannins may be divided into the condensed facilitate pollination and aid dispersal of seeds
tannins (proanthocyanidins), which are com- in coloured fruits. The red, purple and blue
plex and not readily hydrolysed, and the colours of most flowers, and red colours of most
hydrolysable tannins which consist of simple fruits and autumn leaves, are due to antho-
Table 5.1 Percentage composition of lignin from different sources
Source Coniferyl alcohol Sinapyl alcohol p-Hydroxycinnamyl

(guaiacyl group) (syringyl group) alcohol
Softwoods 80 6 14
Hardwoods 56 40 4
Grasses 44 34 22
Phenolics 59
CH 20H CH 20H CH 20H

I I I
CH CH CH
II II II
CH CH CH
QOCH'OH
OCH 3 /QOCH' OH
0 OH
coniferyl alcohol sinapyl alcohol p-hydroxycinnamyl

alcohol
Figure 5.6 The structure of lignin precursors and typical components of lignin: coniferyl alcohol; sinapyl
alcohol; p-hydroxycinnamyl alcohol.
are l3-glucosides of anthocyanidins. The com-
monest are indicated in Table 5.2.
A sugar is normally present at the 3 and
sometimes the 5 position, and some of these
may be di- or trisaccharides. The colour is
OH influenced by the chemical structure - gener-
ally methylating OH groups and glycosylation
gallic acid have a reddening effect. pH also has a strong
effect on colour: pelargonidin is red in acid
solution but blue in alkaline. The colour is also
altered by the presence of co-pigments such as
flavone-glucosides and hydrolysable tannins,
which intensify the colour and shift it towards
HO the blue. The presence of metal ions such as
Fe2 + or AP+ also influence colour. Thus the
colour of both blue cornflowers and red roses
is due to cyanidin derivatives, but in corn-
flower these are complexed with iron and
OH flavones.
OH Flavones
These exist as glycosides of flavones and
flavonols which are mostly yellow and cream.
OH The basic structure of flavones and flavonols is
shown in Figure 5.S. They may contribute
example of condensed tannin from body to white or cream flower colours and act
sorghum
as co-pigments. They absorb light strongly in
Figure 5.7 The structure of gallic acid and a con- the UV which may help them attract insects
densed tannin from sorghum. and prevents damage to the photosynthetic
apparatus from excess light.
cyanins. (Yellow and orange colours as in toma-
5.5 PEPTIDE BONDS
toes and some yellow flowers tend to be due to
carotenoids.) In the cell anthocyanins are con- During the formation of proteins, amino
centrated in the vacuoles (not in the plastids as acids are joined together by condensation
carotenoids are). Chemically the anthocyanins reactions to form long chains (polymers).
Table 5.2 Properties of some anthocyanins
Anthocyanin Colour Typical source R j Rz

Pelargonidin Red Geranium H H
Delphinidin Blue Delphinium OH OH
Cyanidin Red or blue Ripe apples, cornflower OH H
Petunidin Purple Petunia OCH 3 OH
Peonidin Reddish Peony OCH 3 H
Malvidin Mauve Mallows OCH3 OCH 3
Protein function and structure 61
OH
Anthocyanin ring structure
o o
a flavonol a flavone
Figure 5.8 The structure of some flavonoids: anthocyanin ring structure; a flavonol; a flavone.
Substitution of various groups at RJ and ~ in the anthocyanin ring gives rise to pigments with a wide
range of colours (see Table 5.2).
These chains of amino acids are known as An example is the pentapeptide enkephalin
peptides and the bonds that join them are which is produced and used in the brain as a
peptide bonds. The structure of a peptide natural pain-killer.
bond is shown in Figure 5.9.
The groups marked ~ and Rz are the side
chains of the amino acids. This compound is 5.6 PROTEIN FUNCTION AND STRUCTURE
an example of a dipeptide: it consists of two Proteins are polymers of amino acids linked
amino acids, but other amino acids can be together in straight chains to form polypep-
added to extend the chain. Some small peptides. They consist of between approximately
tides are very important compounds in their 100 and 3000 amino acid residues. As the aver-
own right and have strong biological activity. age molecular weight of an amino acid is
about 110, protein molecular weights vary
between about 10 000 and 300 000.
The functions of proteins are very varied.
They may act as enzymes, as means of storing
nitrogen in a biologically accessible form, as
structural components of cells, etc. The unique
property that makes them particularly impor-
peptide tant is their ability to fold into well-defined
bond three-dimensional shapes or conformations so
Figure 5.9 The structure of a peptide bond that they can bind very strongly to other mol-
between two amino acids. ecules. This gives them the ability to 'recog-
nise' other molecules in an extremely specific structure of a very simple protein, bovine
way. Thus enzymes can recognise their sub- insulin. This has two polypeptide chains
strates, antibodies can recognise the corre- which are linked together by three disulphide
sponding antigen, etc. Even very small bridges. Of these bridges one (A) is within a
changes in the shape of proteins usually pre- single chain whereas the others, (B) and (C),
vent them from carrying out their normal link the two chains.
functions correctly.
The structure of proteins can be described
5.6.2 SECONDARY PROTEIN STRUCTURE
at several levels, as summarized in Table 5.3.
Each of the peptide bonds in a peptide chain
can rotate so that a long chain would have a
5.6.1 PRIMARY PROTEIN STRUCTURE
very flexible and bendable structure. It
This is the sequence in which amino acids are achieves a fairly rigid structure because of the
linked to one another by peptide bonds. As we formation of hydrogen bonds between atoms
shall see when we come to look at the way in within the chains. In a hydrogen bond, a
which a protein is synthesized, the order of hydrogen atom essentially becomes shared
the amino acids is 'written down' in the genet- between two other atoms, e.g. the H of an NH2
ic material of the cell. can be shared between its own N atom and the
At one end of the chain there is a free amino o atom of a c=o group. Although individual
group (N-terminal) and at the other a carboxyl hydrogen bonds are really quite weak, there
group (C-terminal). These groups normally are so many of them that they hold the
carry a positive and negative charge, respec- polypeptide chain very firmly in shape.
tively. Some of the side chains (or R groups) Examples of the formation of such hydrogen
also carry positive or negative charges. These bonds are shown in Figure 5.12.
features of the primary structure of a typical A number of distinct arrangements of the
protein are shown in Figure 5.10. polypeptide chain occur commonly in pro-
There is a further type of covalent bond that teins. In some cases hydrogen bonding takes
occurs in proteins. This is the disulphide place between groups which are close togeth-
bridge formed between cysteine side chains er in the same polypeptide chain, resulting in
on the same or neighbouring chains. The the chain being twisted into a helix, which is
cross-linked, double amino acid formed in this usually called the a-helix. In this structure
way is called cystine. Figure 5.11 shows the there are 3.6 amino acids for each turn of the
Table 5.3 Levels of organization in the structure of a protein
Protein structure Organization
Primary The sequence of amino acids in the polypeptide chain or chains. The chemical bonds
which maintain the primary structure are covalent ones (peptide bonds).
Secondary Repeating structures recognizable within the 3-D structure of the polypeptide chains.
There are several types of secondary structure: a-helix; J3-pleated sheet; triple helix, etc.
Secondary structure is maintained largely by hydrogen bonds formed between the
atoms of different peptide bonds.
Tertiary The overall 3-D shape of the polypeptide chain describing its bending, twisting and
meandering in space. This is maintained by large numbers of weak bonds, e.g. hydro-
gen bonds, hydrophobic bonds, ionic bonds, together with covalent disulphide
bridges.
Quaternary The way in which a number of polypeptide chains, each with its own tertiary struc-
ture, come together, perhaps with other molecules, to form the final, active protein.
Protein function and structure 63
..-_ .. _--------- ...
. .
H H H 0: H H H 0 H
I I I II: I : I I II I _
NH3+- C - C - N - C - C -7 N - C - C :- N - C - C - N - C - CO 2
I II I :I I II: I I I
R1 0 R2 :H Rx 0: Ry H Rz
................ : n
amino terminal end carboxyl terminal end

N-terminal end C-terminal End
Figure 5.10 The primary structure of a protein. The chai~ has ~n amino (N-terminal) end and a carboxyl
(C-terminal) end, and normally contains 100 or more ammo aCIds.
helix and each turn occupies about 0.54 nm 5.6.3 TERTIARY STRUCTURE
along the length of the chain. The hydrogen
The tertiary structure describes how the
bonds between peptide groups lie parallel to
polypeptide backbone of the protein is bent
the peptide chain (Figure 5.13).. . and twisted. Much of this is determined by the
Some proteins such as a-keratm, found m
nature of the amino-acid side chains and
hair, wool and hooves, consist almost entirely
whether they are hydrophilic or hydrophobic.
of amino acids arranged in the form of an a-
In biological systems most proteins exist in
helix. Helical chains may aggregate to form
solution or suspension in water, and the pres-
long, twisted fibres which give the struct~res
ence of this water has a great influence on the
their great strength. In most globular protems,
shape of the protein. The protein chain will
however, only parts of the peptide chain exist
arrange itself so that as many hydrophobic
as a-helix whilst others have different confor-
groups as possible point towards the middle. ~f
mations.
the protein structure, whilst the hydrophIlIc
Another type of secondary protein struc-
ones point towards the surrounding water.
ture is called the l3-pleated sheet. In this struc-
This is a stable structure as in the middle of the
ture several individual peptide chains, laid
molecules the non-polar groups can interact
side-by-side, are held together by hydroge~
with one another and are kept away from the
bonds between the peptide groups. ThIS
water, whilst the polar groups on the outside of
results in a very strong structure, rather like
the molecule are stabilized by interaction with
corrugated cardboard. The peptide chains
water molecules. Any change in the nature of
involved may run in the same direction (paral-
the charges on the side chains (for instance by
lel pleated sheet) or in opposite directions
changing the pH of the solution) will alter the
(anti-parallel pleated sheet) and they may be
relationship between the water and the protein.
part of the same, or different, polypeptid.es
As its environment changes the protein will
(Figure 5.13). Silk is an example of a materIal
change its shape to restore the most favourable
consisting of proteins composed mainly of
interactions between the water and the
antiparallel l3-pleated sheets. Most globular
hydrophilic and hydrophobic groups.
proteins contain some regions where the pep-
The tertiary structure is maintained by:
tide chains form a l3-pleated sheet as well as
some which form an a-helix. The difference • hydrogen bonds formed between amino
between a-helix and l3-pleated sheet may be of acid side chains;
great biological significance in prions, which • ionic bonds formed between groups with
are described in section 5.S. opposite charges;
A chain B chain
I I
CH-R1 CH-R2
~H2 ~H2 I I
Gly Phe
C=O····· ·H-N
I
lie
I
Val
I I
I I N-H ...... O=C
Val
I
Asn
I I I
Glu Gin CH-R3 CH-R4
I I
Gin His I I
I I
,cys B LTu Figure 5.12 Hydrogen bonds formed between
components of peptide bonds. Such bonds help to
S Cys - S - S - cys stabilize the secondary structure of the protein.
Ala Gly
A I I
Ser Ser
I I • hydrophobic interactions between hydro-
S Val His phobic amino-acid side chains;
L.. I I
cys LTu • covalent disulphide (-5-5-) bridges between
Ser Val cysteine residues.
I I
Leu Glu Although many of these bonds are individ-
I I
Tyr Ala ually quite weak, their large number stabilizes
the structure of the protein.
Gin Leu
I I
Lyu Tyr 5.6.4 QUATERNARY STRUCTURE
Glu Leu
I I Many proteins do not consist of a single
Asn Val
I C I polypeptide chain; instead they may have sev-
Tyr _S- Cys eral chains or subunits, which may be identical
cys _S ~IY to or different from one another. In addition,
Asn Glu
many proteins have prosthetic groups which
I are not made of amino acids but are essential
Arg for the activity of the protein. Prosthetic
I
Gly groups may be complex carbohydrates, metal
I ions or complex polycyclic compounds such as
Phe
I haem in haemoglobin, myoglobin or
Phe
I cytochromes. In the molecule of haemoglobin
Tyr (Figure 5.14) one haem group is associated
Thr with each of the four subunits and can be
I identified from its flat ring structure.
Pro
I Also regions of the polypeptide where the
Lys chain has taken up a-helix or j3-pleated sheet
Ala structures can be seen. These regions are inter-
spersed with other regions where no regular
Figure 5.11 The primary structure of bovine structures can be seen.
insulin. The protein consists of the A chain (21
amino acids) and the B chain (30 amino acids). Interactions between subunits are important
The chains are linked by two disulphide bridges, in maintaining protein structure. If the rela-
and two cysteine residues in the A chain are tionship between the subunits is changed then
joined by a further disulphide bond. the protein may alter its biological activity.
a-helix ~-pleated sheet
Figure 5.13 Outline structures of a-helix and l3-pleated sheets showing how the structures are stabilized by hydrogen bonds between compo-
nents of the peptide bonds.
Figure 5.14 The structure of haemoglobin. The four haem groups, each associated with one of the sub-
units, can be seen. (Reproduced with permission from Smith, c.A. and Wood, E.J. (1991) Biological
Molecules, 1st edn, Chapman & Hall, London, p. 54.)
Most commonly, biological activity is lost when the N atom of asparagine side chains, and
the subunits dissociate. Some enzymes consist these may play an important part in determin-
of several subunits, and changes in the interac- ing the protein's functions.
tions between them are of great importance in
controlling the activity of the enzyme accord-
5.7 PROPERTIES OF PROTEINS
ing to the needs of the cell (see Chapter 6).
After their synthesis, many proteins are fur- Most proteins are soluble in water or in dilute
ther modified by the action of enzymes. The salt solutions but are denatured by organic sol-
amino acid hydroxyproline, for example, is vents. However, some proteins do dissolve in
found in collagen and is involved in formation organic solvents, and such differences in solu-
of cross-links between adjacent molecules. The bility can be used to divide proteins into classes
amino acid is not incorporated into the protein which show broadly similar behaviour. A sys-
in this form. Instead proline is inserted and is tem of classification of plant proteins based on
subsequently hydroxylated by the action of their solubility in different solvents has been
the enzyme proline hydroxylase which used for almost 100 years. In spite of its age this
requires the presence of ascorbic acid (vitamin system is still often used for the classification of
C) for activity. seed proteins and represents a useful way of
Carbohydrates are also added to many pro- grouping other proteins as well (Table 5.4).
teins by covalent attachment, normally to the This classification is useful because the
o atom of serine or threonine residues or to glutelins and prolamins are deficient in some
Properties of proteins 67
Table 5.4 Classification of proteins on the basis of their solubility
Type of protein Properties Functions
Albumins Soluble in water and in Mainly globular, metabolic

dilute salt solutions proteins; includes most of
the enzymes, etc.
Globulins Insoluble in water but Similar functions to the
soluble in dilute salt albumins, also main type of
solutions storage protein in legumes
and oats
Glutelins Insoluble in water or dilute salt Insoluble enzymes,
but soluble in dilute ribosomal and membrane
acids and bases proteins etc., main storage
protein in rice seeds
Prolamins Insoluble in water, dilute salt Storage proteins in most
or dilute acids but soluble in cereal seeds except for rice
70-90% ethanol and oats
of the essential amino acids, which limits their Ionic strength is defined as:
usefulness for feeding non-ruminant animals.
This point will be discussed in more detail in
Chapter 23.
The properties of albumins and globulins where ci == the concentration of the ith ion
are rather similar and the distinction between and Zi == the charge of the ith ion.
them is often not very clear-cut. These types of Thus salts containing multivalent ions have
proteins have the widest distribution and they a greater influence on the solubility of proteins
have the properties which are normally than those containing only monovalent ones.
thought of as being typical. The remaining Because of the effect of ionic strength on pro-
parts of this section discuss the properties of teins, they are usually studied when dissolved
these groups. The solubility of most proteins is in dilute salt solutions under conditions similar
influenced by a number of factors which are to those which exist in the cell. Most enzymes
discussed below. usually work best under these conditions.
Salting out is a very mild process and is
often employed to precipitate proteins from
5.7.1 IONIC STRENGTH AND PRESENCE OF solution without damaging them. This may be
SPECIFIC IONS used as a means of concentrating the protein
Most proteins are not very soluble in pure or when separating one protein from another.
water but their solubility is usually increased Ammonium sulphate is often used as the salt
by the addition of small amounts of some inor- in these processes because of its high solubili-
ganic salts. However if larger amounts of these ty in water and its ability to dissociate into
salts are added then the solubility of the pro- highly charged ions.
teins may be decreased again. These processes Proteins carry charges in solution and, at
are known respectively as 'salting in' and 'salt- alkaline pHs, they have a net negative charge
ing out' (Figure 5.15a). The important factor and therefore behave as anions. In the pres-
which determines the solubility of the protein ence of many metal ions, particularly those
is the ionic strength of the solution rather than with valencies greater than one, proteins will
the concentration of the salt. coagulate. Heavy metal ions such as Cd +, Cu2 +,
3
0.8
0.6 ....... 2.5

I
E
0.4 z 2
~
:a:::I .s>-
CI
0.2
0 1.5
C/)
0 Isoelectric
CI ==
:a point
0 :::I 1
- -0.2 0
-0.4
If)
0.5 ~
-0.6 0 0
0.5 1.5 2 2.5 3 4.5 5 5.5 6
Ionic strength pH
(8) (b)
Figure 5.15 (a) The effect of ionic strength on solubility of a typical protein. At low concentrations salts
normally increase the solubility (salting-in) but at higher concentrations they cause precipitation (salting-
out). (b) The effect of pH on the solubility of typical proteins. Minimum solubility occurs at the isoelectric
point.
Fe3+, Hg2+, Pb2+ and ZnZ+ all cause proteins to side chains. As the solution is made more
precipitate. This accounts for the toxic nature acidic the net charge becomes positive, and as
of most of these metals if they enter the food it is made more alkaline the net charge
chain or are accidentally included in the diets becomes more negative. At some point the
for animals. Some (e.g. Hgz+ and Pb2+) combine number of negative charges on the protein
specifically with -SH groups in the proteins, due to ionized acid groups (-COO·) and the
which may be essential for enzyme activity. number of positive charges on the basic amino
At acidic pH levels, protein molecules carry acid groups (-NH3 + or = NHz+) are equal.
a net positive charge. Under these conditions When this point, called the isoelectric point
many anions will cause proteins to precipitate. (pI), is reached, the solubility of the protein is
The anions from some acids are very effective at its lowest and many proteins precipitate
at this, for example trichloroacetic acid (TCA) from solution under these conditions (Figure
is frequently used in analysis to precipitate all 5.15b). This is because repulsion between indi-
proteins before determination of some other vidual protein molecules is minimized at this
analyte. Under natural conditions tannins, pH so that they can aggregate and precipitate.
found in many plant materials, will precipitate The effect is demonstrated· very easily by
proteins. This phenomenon occurs naturally adding lemon juice to milk. The proteins will
in animal feeds which contain high levels of precipitate as the pH falls to about 4 which is
tannins and it may reduce the availability of the isoelectric point of casein, the most abun-
proteins in the diet. dant protein in milk. This process can be used
for the commercial preparation of casein and
occurs during the preparation of some milk
5.7.2 EFFECT OF pH
products such as cottage cheese.
As the pH is altered the number of charges on
a protein molecule in solution changes. This
5.7.3 DENATURATION
results from changes in the extent of ioniza-
tion of the N- and C-terminal groups at the The addition of ammonium sulphate, or
ends of the polypeptide and of groups in the adjusting the pH to the isoelectric point, usu-
Prions 69
ally results in precipitation of proteins. marked in the early stages by intense itching
However, removing the salt or adjusting the and trembling. As it develops over a period of
pH again often allows the proteins to redis- 1.5-6 months, walking is impaired and in the
solve. Under some circumstances precipitation final stages the animal is unable to rise.
is irreversible and the proteins become dena- Microscopic examination of the brain tissue
tured. Denaturation occurs when a protein showed a sponge-like structure with large
uncoils and loses its three-dimensional shape. plaques of protein. Related conditions were
When this happens, the non-polar groups, described in humans (Creutzfeld-Jacob
which are usually folded into the centre of the Syndrome and Gerstmann-Strussler
protein molecule, are exposed to the water Syndrome) and in mink, deer and cats. The
surrounding it. This is an unstable arrange- conditions were however quite rare. The dis-
ment and it is usual for protein molecules to eases are known in general as transmissible
coalesce so that the non-polar groups of one spongiform encephalopathies (TSEs).
can interact with those of another. Heating, In the 1980s, particularly in Britain, there
extremes of pH, addition of organic solvents or appeared a bovine version of the disease
addition of detergents may cause denatura- (bovine spongiform encephalopathy - BSE,
tion. sometimes called mad cow disease). From a
few sporadic cases in the earlier part of the
1980s, the incidence had risen to hundreds of
5.7.4 EFFECT OF HEAT
thousands of cases by the early 1990s. The
Heating proteins usually has drastic effects on blame was laid on the practice of feeding meat
their biological activity. As they are heated the and bone meal to cattle and to a change in the
atoms vibrate more rapidly and the carefully conditions used for its manufacture. Until the
arranged secondary, tertiary and quaternary early 1980s solvents were used to extract as
structures are lost. The chains usually assume much fat as possible from the meal because
more randomly arranged structures with this was a high-value product. The last traces
fewer charged groups to the outside of the of organic solvent were removed by using
molecules, which greatly reduces their solubil- superheated steam. With a drop in the world
ity. The effects of such denaturation can read- price of oils and fats there was no longer any
ily be seen during cooking of protein-rich incentive to remove it all from the meat and
foods such as eggs. bone meal, and this step was dropped from
Denaturation is not always undesirable. the processing.
Cooking proteins may make them more sus- It has been known for many years that the
ceptible to attack by enzymes and thus hasten causative agent of scrapie was neither a bacteri-
their digestion in animals. It can also be useful um nor a virus, and a suggestion was made in
to denature proteins which are potentially the 1960s that it was caused by a protein free of
harmful such as some enzyme inhibitors dis- nucleic acids. The protein was extremely resis-
cussed in Chapter 28. tant to denaturation, surviving all but the most
extreme physical conditions. Similar proteins
were then shown to be associated with the
5.S PRIONS
related degenerative conditions in man, mink
Evidence is accumulating for an important role and deer. The name prions has been given to
of some proteins in the determination of their these proteins. Further research has shown
own three-dimensional structure. The disease them to be a modified form of a glycoprotein
scrapie has been known for many years to that occurs as a normal component of nerve
cause severe nervous disorders in sheep and, membranes. It is rooted in the membrane
more rarely, goats. It is a progressive disease through a phosphoinositol group. These pro-
teins are known as prion proteins (PrP), the one However, the two proteins differ from one
which causes scrapie is known as Prpsc where- another in their sensitivity to proteolytic
as the normal cellular one is PrPC. The two pro- enzymes, in the conformation of their
teins have the same polypeptide sequence polypeptide chains (PrPC has 42% a-helix and
coded by the same DNA sequence of the gene. 3% l3-pleated sheet, but PrPSc has 30% helix
In sheep the immune system fails to give any and 43% pleated sheet) and in their stability in
protection against scrapie. This is explained by the cell as well as in their capacity to transmit
the fact that the causative agent is actually a spongiform encephalopathy.
variant of a quite normal protein that is not in The exact way in which the disease is
any way foreign to the animal. Their molecular spread is not clear, however a suggested way
weights are identical at around 34 kDa. In both is shown in Figure 5.16. In this theory the
cases there is a considerable degree of post- infective PrpSc protein reacts with a normal
translational modification of the polypeptide molecule of PrPC to form a dimer. This induces
by the addition of seemingly identical carbohy- the Prpc to change shape so it is in the same
drate side chains fixed to an asparagine group conformation as Prpsc. The dimer now sepa-
and a glycolipid which contains phosphoinosi- rates to release two molecules of PrPSc which
tol near the C-terminal end of the chain. are free to transform more PrPC.
PrP Sc seed
Dimeric form
PrP Sc
Figure 5.16 Proposed means of multiplication of prion proteins. Interaction of infective PrPS<: (mainly 13-
pleated sheet) causes normal Prpe (mainly a-helix) to change its conformation to that of the infective form.
ENZYMES 6
6.1 Introduction 71
6.2 Types of reactions catalysed by enzymes 71
6.3 Mode of action of enzymes 72
6.4 Factors contributing to enzyme activity 73
6.4.1 Proximity of substrates at the active site 73
6.4.2 Environment 74
6.4.3 Acid-base catalysis 74
6.4.4 Effects on the stability of substrates and 74
reaction intermediates
6.4.5 Formation of covalent enzyme-substrate 74
intermediates
6.5 Factors affecting the rates of enzyme- 76
catalysed reactions
6.5.1 Enzyme concentration 76
6.5.2 Substrate concentration 76
6.5.3 Temperature 79
6.5.4 pH 79
6.5.5 Presence of inhibitors 81
6.5.6 Presence of coenzymes 84
6.6 Allosteric enzymes 90
6.7 Molecular recognition 91
6.7.1 Receptors 91
6.7.2 Antibodies 91
6.1 INTRODUCTION • They change the speed of reactions, but

Enzymes are molecules which catalyse bio- cannot make reactions occur which would
chemical reactions. They act on their sub- not otherwise take place. Thus they change
strates and convert them into products. the rates but not the equilibrium constants
Although a few examples of catalytic RNA of reactions.
molecules are known these are quite unusual, • They are highly specific. This means that
and the vast majority of biological catalysts are they will act only on substrates which have a
proteins. particular structure. They convert substrates
Enzymes have a number of particularly into products with almost 100% efficiency.
interesting and important properties.
6.2 TYPES OF REACTIONS CATALYSED BY
• They bring about changes in their sub-
ENZYMES
strates without being changed themselves,
i.e. they are true catalysts. Although thousands of different reactions are
• They increase the rates of reactions many catalysed by enzymes they can be classified
thousands or even millions of times. into six basic types (Table 6.1). As we study
72 Enzymes
metabolism we shall see examples of all of ture of the proteins themselves - ionic bond-
these. ing, hydrophobic bonding, hydrogen bond-
ing, etc. (see Chapter 5). The binding of sub-
strate to the enzyme is a reversible process and
6.3 MODE OF ACTION OF ENZYMES
the substrate is free to dissociate from the
Enzymes function by lowering the activation enzyme-substrate complex again.
energy of reactions. They do this by providing Many enzymes do not use a single sub-
an easy route for the reaction. This usually strate, but instead bring about a reaction
occurs because they allow a reaction to pro- between two or more substrates, all of which
ceed via several small steps instead of one have, at some time, to bind to the enzyme. At
large one. the active site reactions take place between the
When molecules react together they do so substrates and they are converted into prod-
by forming an unstable intermediate called a ucts which then dissociate from the enzyme,
transition state, which has a higher energy freeing the enzyme to bind more substrate.
content than the original reactants. The forma- This may be represented in the following
tion of this intermediate therefore represents way:
an energy barrier through which the mole-
cules must pass, and the rate of the reaction is
E+S ~ ES -> E+P Equation 6.1
determined by the number of molecules with
kz
sufficient energy to pass over the barrier. An
enzyme may function by lowering this activa- The active site usually takes the form of a
tion energy barrier, as shown in Figure 6.l. cleft in the surface of the enzyme. This cleft is
Notice that the overall free energy change for lined by amino acids from different parts of
the reaction (difference between the energy of the polypeptide chain which has been folded
the reactants and products) is unaltered by the to form the site. It thus involves amino acids
presence of the enzyme. which are distant from one another in the pri-
The first step in any enzyme-catalysed reac- mary amino-acid sequence but which come
tion is for the enzyme to bind its substrate or close together in space. For this reason
substrates to form an enzyme-substrate com- enzymes are very susceptible to conditions
plex (ES). Groups of atoms on the surface of which cause even small changes in their three-
the enzyme molecule interact with specific dimensional structure, as this distorts the criti-
parts of the substrate molecule. The part of the cal structure of the active site.
enzyme at which the substrates are bound is There are usually many points of attach-
called the active site (Figure 6.2). Binding of ment between the substrate and the surface of
the substrate to the enzyme is accomplished the enzyme protein. The substrate must fit the
through a combination of many of the same active site very precisely if it is to interact with
forces which are used to maintain the struc- all of these attachment sites. This is one of the
Table 6.1 Types of enzyme-catalysed reactions
Enzyme Type of reaction
Oxidored uctases Catalyse oxidation and reduction reactions

Transferases Transfer groups from one molecule to another
Hydrolases Hydrolyse bonds by the' addition' of water
Lyases Catalyse the removal of groups from substrate molecules without hydrolysis
Isomerases Catalyse the interchange of optical or structural isomers
Ligases Catalyse the linking together of two or more molecules
Factors contributing to enzyme activity 73
Reaction c~ordinate
Figure 6.1 Reaction profile for (a) uncatalysed and (b) catalysed reactions. The activation energy jor the
catalysed reaction (.:1C·) is less that that for the uncatalysed reaction(.:1C·J. Hence more molecules can
cross the energy barrier and the reaction is accelerated. Note that the overall free energy change for the
reaction (.:1CO) is unchanged.
factors which allows enzymes to be so specific The idea that the shape of an enzyme's
in binding their substrates and even to distin- active site is complementary to that of the sub-
guish between optical isomers of the same strate is called the lock and key model and it is
compound. a useful way of looking at the relationship
between enzymes and substrates. However in
many cases it seems that enzymes may actual-
ly fold themselves around their substrates
making an even better fit - this is called the
induced fit model.
6.4 FACTORS CONTRIBUTING TO ENZYME

ACTIVITY
Although each individual enzyme has its own

particular mechanism, the catalytic efficiency
of most enzymes is influenced by a number of
common factors.
Figure 6.2 The enzyme hexokinase and its sub- 6.4.1 PROXIMITY OF SUBSTRATES AT THE
strate glucose. The substrate is small in compari- ACTIVE SITE
son with the enzyme, and binds to the enzyme at
the active site which lies in a cleft in the enzyme Although it is common to talk about an
surface. (Reproduced with permission from Smith, enzyme and its substrate, most enzymes actu-
c.A. and Wood, E.J. (1991) Biological Molecules, 1st ally catalyse the reaction between two or more
edn, Chapman & Hall, London, p. 109.) substrates. One of the functions of an enzyme
74 Enzymes
is to bind the substrates in such a way that the 6.4.5 FORMATION OF COVALENT
groups which will react are close together and ENZYME-SUBSTRATE INTERMEDIATES
have the correct orientation. This greatly
increases the rates at which they react and is The side chains of some amino acids at the
called the proximity effect. active sites of enzymes can form covalent
bonds with groups within the bound sub-
strates. The CH2-OH group of serine is one
6.4.2 ENVIRONMENT
such group which is present at the active site of
The environment in the immediate vicinity of a number of proteolytic (protein hydrolysing)
the active site of many enzymes is very differ- enzymes such as trypsin and chymotrypsin.
ent from that found in the aqueous solutions The CH2-OH group attacks the peptide bond
surrounding the enzyme. For example the and the acyl group becomes covalently
active site is often lined by the side chains of attached to the CH 2-O- group (Figure 6.3).
non-polar amino acids which may greatly This intermediate then breaks down to release
influence the rates at which many chemical the acid and regenerate enzyme.
processes take place. Another series of enzymes, which includes
proteolytic plant enzymes such as papain and
bromelain, use the side chain of cysteine
6.4.3 ACID-BASE CATALYSIS (-CH 2-SH) in a similar way. These enzymes
Enzymes can make some atoms or functional are called the cysteine proteases. In yet anoth-
groups in their substrates more reactive by er group of enzymes, using ATP as a substrate,
adding a proton or removing a proton from the side-chain carboxyl groups of aspartate or
them. This is known as acid-base catalysis. glutamate take part in similar reactions.
Usually this is the function of the side chain of Metal ions contained within enzymes may
one specific amino acid at the active site. destabilize substrates. In carboxypeptidase a
Groups such as the side-chain carboxyl groups zinc atom polarizes the carbonyl group of the
of aspartic or glutamic acids, or the imidazole peptide bond of the substrate and thereby ren-
ring of histidine, can function in this way. ders the peptide bond more easily broken.
Depending on the pH they may be able to In many enzymes, several of these effects
function as either acids or bases. may operate at once. For example in chy-
motrypsin and papain, not only does the serine
or cysteine group attack the substrate molecule,
6.4.4 EFFECTS ON THE STABILITY OF but a nearby histidine residue also acts as an
SUBSTRATES AND REACTION INTERMEDIATES acid-base catalyst. The presence of this histi-
dine results in the serine or cysteine residue at
Binding of substrates to enzymes may create the active site of these enzymes being very
strains in the bonds between atoms of the sub- much more reactive than similar residues
strates. This occurs because some distortion of found in other parts of the protein.
the substrate may take place as it binds to the The net result of all these processes taking
active site. In other enzymes, reaction interme- place at the active site is that substrates are
diates or transition states may be stabilized by converted into products which then dissociate
spreading the charges on them as a result of from the enzyme, leaving it free to act on fur-
their interaction with the enzyme. ther substrate molecules. The speed at which
Destabilizing the substrate and stabilizing products are formed varies enormously from
reaction intermediates both decrease the acti- enzyme to enzyme. It can be represented by
vation energy barrier and accelerate conver- the turnover number of the enzyme, which is
sion of substrates into products. the maximum number of substrate molecules
Factors contributing to enzyme activity 75
His
I / \ I
C-Q' HN ~N ""' H-O
I
o
I
C-QH··· N ~N-H
I
o
le·
I
o
Figure 6.3 Mechanism of chymotrypsin action. Chymotrypsin hydrolyses peptide bonds. The reaction
involves transfer of the acyl (R-CO) group of the substrate to a serine residue (at position 195 in the pri-
mary sequence) at the active site to form an acyl-enzyme intermediate which then breaks down to
release the acid and regenerate free enzyme. The aspartic acid residue (position 102) and histidine
residue (position 57), also at the active site, activate the serine and speed up both formation and break-
down of the acyl-enzyme by acid-base catalysis.
76 Enzymes
acted on by one enzyme molecule each sec- 6.5.2 SUBSTRATE CONCENTRATION
ond. This varies from about 0.5 S-1 for
The dependence of Vo on substrate concentra-
lysozyme to about 4 x 107 S-1 for catalase.
tion [5] for most enzymes is shown in Figure
6.4. As the substrate concentration is raised, Vo
6.5 FACTORS AFFECTING THE RATES OF increases but the rate of increase becomes less
ENZYME-CATALYSED REACTIONS until increasing substrate concentration has no
further effect on vo' At low substrate concentra-
The rates at which enzymes convert their sub-
tions Vo is proportional to [5] but at high sub-
strates into products depend on many factors.
strate concentrations it is independent of [5].
Their study is called enzyme kinetics and is
The shape of this curve can be understood
important because enzymes determine the
by referring to Equation 6.1. At low substrate
rates at which most vital processes occur in all
concentrations, increasing [5] causes more
living organisms.
enzyme-substrate complex (ES) to form, and
When a substrate is added to an enzyme,
the rate of formation of product (P) increases.
the enzyme begins to convert it into products.
At high substrate concentrations the active
Enzymes also catalyse conversion of the prod-
sites of the enzyme become saturated by sub-
ucts'back into substrates. Eventually the reac-
strate molecules. This means that substrate is
tion will reach equilibrium when products are
bound to the active site of every enzyme mol-
converted back into substrates at the same
ecule (in terms of Equation 6.1 all E is convert-
rate as they are formed. The position of the
ed to ES). Under these conditions, adding
equilibrium in these enzyme-catalysed reac-
more substrate cannot increase the rate of the
tions is no different from that of the uncatal-
reaction any further as there is no free enzyme
ysed reaction.
to which it can bind.
The rate at which product is formed when
The shape of the plot of Vo against [5] is a
an enzyme and substrate are first mixed hyperbola and it can be described mathemati-
together is called the initial rate of the reac-
cally by the following equation. This is the
tion (va) and it depends on many factors Michaelis-Menten equation and KM is the
including:
Michaelis constant.
• enzyme concentration
• substrate concentration
Equation 6.2
• temperature
• pH
• presence of inhibitors The terms V max and KM determine the
• presence of co-factors. shape of the curve. V max is the maximum rate
These will be considered in turn. that the reaction can reach at high substrate
concentrations. Under these conditions the
enzyme is said to be saturated with substrate
6.5.1 ENZYME CONCENTRATION
and the active sites of all of the enzyme mol-
Under normal circumstances the initial rate of ecules are occupied by substrate. Adding
reaction (va) is directly proportional to the more substrate cannot increase the rate of the
concentration of enzyme present. This allows reaction any further. V max therefore tells us
enzyme activity to be determined by measur- something about k3 (see Equation 6.1), i.e.
ing vo' This parameter is often used in the about how efficiently the enzyme converts ES
diagnosis of human and animal diseases as a into P; the larger the value of V max the more
measurement of enzyme activity in tissue or rapidly the enzyme will convert substrates to
blood samples. products.
Factors affecting the rates of enzyme-catalysed reactions 77
100
90
80
70
60
>0 50
40
30
20
10
O+-~--~---+----~----+---~
4 6 8 10
IS]
Figure 6.4 Dependence of rate of enzyme-catalysed reaction (vo) on substrate concentration [5]. At low
substrate concentrations Vo is proportional to [5] but at high substrate concentrations Vo becomes inde-
pendent of [5]. Under these conditions the enzyme is 'saturated' by substrate. The shape of the plot is
given by the Michaelis-Menten equation: Vo = Vrnax / (KM/5+ 1).
KM is defined as the substrate concentration strongly bound substrate, whilst a high value
needed to achieve half of the maximum rate of denotes a weakly bound one.
reaction and it thus has the same units as sub- It should be noted that not all enzymes
strate concentration. It can be shown to be obey Michaelis-Menten kinetics. For some
given by Equation 6.3. complex, allosteric enzymes, the plot of Vo
against [5] is not hyperbolic but sigmoid.
(k z +k3) To determine KM and Vrnax in practice it is
KM=..:......::_..c:..:... Equation 6.3
kj necessary to measl,lre the initial rates of an
enzyme-catalysed reaction (vo) at differing sub-
where k1' kz and k3 are rate constants for strate concentrations [5]. These should be cho-
each step as shown in Equation 6.l. sen so that they range from about 0.5---10 KM . It
The binding of substrates to enzymes is a is also necessary to decide the way in which
reversible process. Once the substrate is the initial rates of reaction will be measured.
bound to the enzyme it may dissociate again One way is to mix enzyme and substrate
or it may be converted into products by the together and, after fixed times, to stop the reac-
enzyme. The attachment of substrate to the tion and determine the quantities of substrates
active site and its dissociation again are usual- or products present. Any suitable method
ly very much faster than the rate at which ES (high-performance liquid chromatography
is converted into P. For many enzymes, there- (HPLC), radiochemical methods, etc.) can be
fore, k2 and k1 are much greater than k3 • Under used to analyse the mixture. However this
these circumstances k3 is very much smaller approach is slow and laborious, and it is much
than kz and can be ignored, thus KM approxi- more convenient to monitor the course of the
mates to k/k j • This is the dissociation constant reaction continuously. For some reactions this
for the enzyme-substrate complex and indi- is very easy as conversion of substrate into
cates how tightly the substrate is bound to the product may result in a change in a property,
enzyme. A low value for KM generally means a such as the absorption of light, which can be
78 Enzymes
measured easily and non-destructively. A par- Measurement of enzyme activity has many
ticularly useful application of this type of direct, practical applications, for example in
method follows the conversion of the coen- diagnosis of disease in humans and farm ani-
zyme NAD+ to NADH or vice versa, which is mals, or in identification of plants for particu-
described in more detail in Section 6.5.6. lar uses. Some enzymes are restricted to par-
Once the values of Vo at different values of ticular locations in normal healthy animals,
[51 have been measured they can be analysed and only appear in other body parts in dis-
to determine KM and Vmax. This can be done eases. Thus the transaminase enzymes are
using commercial computer programs or by normally restricted to liver and muscle tissues
transforming the data into reciprocal form and and creatine kinase to muscle, and the appear-
analysing it in the form of Lineweaver-Burk or ance of high levels of these in blood is an indi-
Eadie-Hofstee plots (Figure 6.5). cation of some disorder. As blood analysis is
0.25
(a)
1Nrrax
-5 25
Figure 6.5 Reciprocal plots are used to examine the dependence of Vo on [5]. (a) Lineweaver-Burk plot:
l/vo is plotted against 1/5. The intercept on the x axis is 1/Vmax and that on the y axis is -1/KM· (b)
Eadie-Hofstee plot: Vo is plotted against viS. The intercept on the y axis is Vmax and the line has a slope of
-KM The Lineweaver-Burk plot gives a poor representation of experimental errors but is a convenient
way of examining the nature of enzyme inhibitors.
easily performed it provides a convenient and quent collisions of reactant molecules; but the
sensitive diagnostic tool in both human and rate is also decreased due to enzyme denatura-
veterinary medicine. Other examples are tion. In general, at low temperatures the former
given in Table 6.2. is more important, so that the rate increases
Measurement of enzyme activity is also until denaturation becomes more important,
often required in the food industry. As exam- when the rate decreases often in quite an
ples, alkaline phosphatase activity has been abrupt way. A temperature is seen at which the
used for many years as a measure of the com- enzyme appears to be working at its fastest.
pleteness of pasteurization of milk, and deter- This is sometimes called the optimum tempera-
mination of the activity of specific enzymes in ture of the enzyme. However it does not have a
feeds can be used as a measure of their quali- unique value: increases in rate occur almost
ty. Soyabeans contain a protein which inhibits instantaneously as the temperature is raised but
the activity of digestive proteolytic enzymes, denaturation may be a relatively slow process.
and also contain the enzyme urease. Heating Thus the optimum temperature measured
the beans denatures both of these proteins. depends on the length of time that the enzyme
Measurement of urease activity can therefore is held at the temperature before its activity is
be used to determine the effectiveness of heat- measured. This is illustrated in Figure 6.6a.
ing in preparation of soyabean meal. In many instances it is found that enzymes
work best at the temperature of the environ-
ment in which the organism is usually found.
6.5.3 TEMPERATURE
Thus in many warm-blooded animals the opti-
The rate of chemical reactions increases as tem- mum temperature of many enzymes is close to
perature increases. The way in which this takes the body temperature, whilst thermophilic
place is described by the Arrhenius equation: bacteria found in hot springs have enzymes
which retain activity above 90°.
E
In(k) = cons tan t - - Equation 6.4
RT 6.5.4 pH
where: k = rate constant; E = energy of acti- Most enzymes function best at a particular pH,
vation; R = gas constant; T = temperature ("K). so that the plot of their activity against pH is a
The rate of a chemical reaction approximate- bell-shaped curve. Some work well over only
ly doubles for each lOoe rise in temperature very narrow ranges whilst others work over a
and those catalysed by enzymes behave just wide range of pHs. Enzymes also differ very
like any other. However, because they are pro- widely in the pHs at which they function best,
teins, enzymes are denatured by high tempera- e.g. pepsin has a pH optimum of about 2, and
tures. As the temperature of an enzyme is alkaline phosphatase about pH 10. Most
raised, two opposing effects take place. The rate enzymes work most rapidly between pH 5
is increased by the greater energy and more fre- and 7 as shown in Figure 6.6b.
Table 6.2 Diagnostic uses for some enzymes
Enzyme Tissue for assay Disorder
a-amylase Serum and urine Pancreatitis

Creatine kinase Serum Disorders of skeletal and cardiac muscle
Alkaline phosphatase Serum Bone diseases, obstructive jaundice
Acid phosphatase Serum Prostatic tumours
Transaminases Serum Liver and muscle diseases
80 Enzymes
increasing
(a) 10
incubation
9 tine
8
..
7
.!! 6
0::
til 5
~
~ 4
0::
3
2
0
0 10 20 30 40 50 60
Terrperature (DC)
120
(b)
100
!! 80
I!
60
~
~ 40
20
0
0 2 4 6 8 10 12 14
pH
Figure 6.6 (a) Dependence of the rates of enzyme-catalysed reactions on temperature. The optimum
temperature is lower when the enzyme activity is measured after being held at the temperature for long
enough for significant denaturation to take place. (b) Dependence of the rates of enzyme-catalysed reac-
tions on pH. Both the optimum pH, and the pH range over which an enzyme is active, vary widely.
Changes in pH alter the number of charges ability of the enzyme to convert enzyme-sub-
on both the substrate and the enzyme, which strate complex to product may also change
alters the ability of groups on the enzyme and with pH for the same reason. Within a certain
substrate to interact with one another by ionic range of pH, on either side of the pH opti-
and hydrogen bonding. Thus there is a pH at mum, the changes which take place are
which interaction of substrate with the active reversible so that activity can be restored by
site is most favourable. On either side of this adjusting the pH back to the optimum.
pH, binding of substrate is less favoured. The However, towards extremes of pH the enzyme
may become denatured, resulting in perma- enzyme by similar interactions. Addition of an
nent loss of catalytic activity. excess of substrate can overcome the inhibi-
tion, so that V max is unaltered but more sub-
strate is needed to achieve the maximum rate
6.5.5 PRESENCE OF INHIBITORS
than in the absence of inhibitor. Thus KM is
Inhibitors are compounds which reduce the increased.
activity of enzymes. Because the interaction of
substrates with the active site of enzymes is
Non-competitive inhibitors
very specific, it can be upset in a variety of
ways. Non-competitive inhibitors bind, not to the
Enzyme inhibitors are divided into classes active site of the enzyme but at other locations,
depending on the way in which they work. and their binding is unaffected by the pres-
The main division is into reversible and irre- ence of substrate. Binding of the inhibitor to
versible inhibitors. In the former case removal the enzyme distorts the active site so that sub-
of the inhibitor from the enzyme restores strate is transformed less effectively by the
activity. In the latter case the inhibitor is so enzyme. Increasing the substrate concentra-
tightly bound, sometimes by formation of tion has no effect on binding of the inhibitor to
covalent bonds, that it cannot be removed. the enzyme. The structure of these inhibitors
may bear no resemblance to that of the sub-
strate as they bind to different parts of the
Reversible inhibitors
enzyme. Their presence can be detected by
These inhibitors become reversibly bound to measuring the rate of reaction in the presence
their enzymes as follows. of increasing concentrations of inhibitor. Vmax
is decreased in the presence of this type of
K;
inhibitor but KM is unchanged.
E+I ~ EI Equation 6.5
Reversible inhibitors are divided into two
Irreversible inhibitors
types.
These become very tightly bound to the
enzyme: usually covalent bonds are formed
Competitive inhibitors
with an amino acid in the protein or with a
Here the inhibitor binds to the active site of the tightly bound coenzyme. The point of attach-
enzyme and, in doing this, prevents the sub- ment is often, but not always, the active site of
strate from binding. The binding of these the enzyme. They may prevent the substrate
inhibitors, however, like that of the substrate, is from binding to the enzyme or prevent the
reversible so that an equilibrium is established. enzyme from converting enzyme-substrate
Either substrate or inhibitor, but not both, may complex into product. Increasing the substrate
be bound at the active site at anyone time. The concentration cannot overcome the inhibition
proportion of enzyme molecules with sub- because there is no competition between the
strate or inhibitor bound will depend on the substrate and inhibitor for binding to the
affinities of the enzyme for substrate and enzyme.
inhibitor and on their relative concentrations
in the solution. Adding more substrate will dis-
Practical applications of enzyme inhibitors
place the inhibitor and overcome the inhibi-
tion. The structure of these inhibitors usually Many enzyme inhibitors have very potent
resembles very closely that of the substrate effects on animals or plants. Some are highly
because they bind to the same part of the toxic and have undesirable effects on a wide
82 Enzymes
range of organisms. Others are more selective
in their action and may be used very effective-
ly in the control of pests and diseases.
Insecticides
:;.
~t:q ~.
'.
1 Direction of tnIveI
of IMIMt impuIIe
Insecticides have allowed many important dis-
=
eases of animals, plants and man to be con-
trolled. Some of the commonest interfere with •• I ~' ...
the function of insect nerves, in which they

O .~-' ~_,qJo
.i. ,' /:
selectively inhibit a particular enzyme «>- , ~ .t,.,;
involved in the transmission of messages. .~ .. «> .
Nerves consist of long fibrous cells (axons) [!] «> «> 0 «>
Postsynaptic
along which electrical impulses are transmitted membrane
by changes in the concentration of ions in the
cell. At the end of each axon the' message' has
to be passed on to the next cell and this is done
at synaptic junctions. The synapse at the end of
the nerve fibre produces a compound called
acetylcholine. This passes to the next nerve
cell, in which it induces changes in the internal
ionic concentration and initiates another nerve
impulse. The acetylcholine produced does not
enter the second cell, but binds to a protein on
the cell surface (Figure 6.7). Once the second Figure 6.7 Transfer of a nerve impulse across a
synaptic junction using acetylcholine. Acetylcholine
cell has responded the acetylcholine must be
(eO) is formed from acetyl-CoA (e) and choline
rapidly destroyed by the initiating cell to stop (0), and is released from the presynaptic membrane
the second cell sending further impulses. The in response to a nerve impulse. Acetyl-CoA stimu-
enzyme that destroys acetylcholine is called lates the postsynaptic membrane and initiates an
acetylcholine esterase. If it is inhibited then impulse before it is broken down by acetyl-
acetylcholine levels increase and nerve impuls- cholinesterase. Inhibition of this enzyme causes
es flow in an uncontrolled way. nerve impulses to flow in an uncontrolled way.
Organophosphorus insecticides function by
reacting with the active site of acetylcholine hyperactivity, convulsions, paralysis and
esterase. They covalently attach a phosphate death) reflect this fact.
group to the side chain of a serine residue at Organophosphorus and carbamate com-
the active site. This has the effect of perma- pounds are toxic to all types of animals, but by
nently and irreversibly inhibiting the enzyme careful selection examples can be found that
(Figure 6.8). are much more harmful to insects than they
The carbamate insecticides have structures are to man. One use of these has been as com-
which are quite similar to those of acetyl- ponents of sheep dip. However, long-term
choline itself and they thus compete for the exposure of farm workers to these compounds
binding sites and act as competitive inhibitors has resulted in serious health problems.
of the esterase. Both these groups of com- Some organophosphorus compounds
pounds stop the nervous system from exerting which are exceptionally toxic to man have
control over muscles, and the symptoms of been made, and used, as nerve gases in chem-
insecticide poisoning of insects (tremors, ical warfare.
F
~ --0 -
r
CH 3 - CH- 0 - CH -CH3
~H3 ~ ~H3
DFP
Figure 6.8 Inhibition of acetylcholine esterase by diisopropyl flu oro phosphate (DFP). DFP reacts with the
-OH group of a serine residue at the active site and becomes covalently attached to the enzyme, irre-
versibly inhibiting its activity.
Herbicides groups of herbicides inhibit the enzyme ace-

A number of herbicides are enzyme inhibitors. toxy acid synthetase, which is required for the
One of these is glyphosate, which acts on synthesis of the branched-chain amino acids
enzymes of a biochemical pathway (the shikim- (Chapter 20). Herbicides which act on this
ic acid pathway) that animals do not have enzyme include the sulphonylureas, imida-
(Chapter 20). In plants and bacteria the aromat- zolinones and the triazolopyrimidines.
ic amino acids (tryptophan, phenylalanine and Like the aromatic amino acids, branched-
tyrosine) are synthesized by this route. Without chain amino acids cannot be synthesized in
these amino acids plants cannot synthesize pro- animals because they lack the enzymes
teins and thus they die. Glyphosate has a struc- required to catalyse the steps in the biosyn-
ture that resembles phosphoenolpyruvate and the tic pathways. The targets on which the her-
competes with it for binding sites on the EPSP bicides act are therefore not present in animals
synthetase enzyme (Figure 6.9). and, unless they have other separate effects,
A number of other herbicides also act as they are normally of low toxicity to man and to
enzyme inhibitors. For example, several animals.
84 Enzymes
COOH COOH
6H 2
6-CH 2
~H 6
6H 2
HO-~-O
HO-~=O 6H
6H Phosphoenol
Glyphosate pyruvate
Figure 6.9 Some herbicides inhibit amino acid biosynthesis. Glyphosate resembles phosphoenolpyruvate
and inhibits the enzyme EPSP synthetase which uses phosphoenolpyruvate as a substrate.
Pharmaceuticals and results from excessive accumulation of

uric acid from the degradation of purines. The
Many very useful pharmaceuticals function by
uric acid crystallizes in joints, which become
inhibitinB' often competitively, specific
extremely painful. It seems to occur because
enzymes in man and animals. Because com-
the enzyme (xanthine oxidase) which converts
petitive inhibitors resemble the substrates for
hypoxanthine into uric acid is particularly
an enzyme, once the specificity of an enzyme
active. Allopurinol closely resembles hypoxan-
is known it is possible to design and synthe-
thine and is used as a substrate by the enzyme
size potential competitive inhibitors. It is
(Figure 6.10). The product however remains
therefore possible to carry out rational design
tightly bound to the enzyme, inhibiting its
of potential drugs.
action and decreasing uric acid production.
PABA and the sulphonamides The com-

pound p-aminobenzoic acid (PABA) is a com- 6.5.6 PRESENCE OF COENZYMES
ponent of the coenzyme folic acid. This is Some enzymes require the presence of small
required for the transfer of groups containing molecules called coenzymes for activity. In
single carbon atoms. Reactions of this type are animals many of these are synthesized from
common and include one of the steps by which vitamins in the diet (see Chapter 8).
the purine nucleotide IMP is synthesized. The
structure of the sulphonamide drugs closely
resembles that of PABA (Figure 6.10). Redox coenzymes (NAD+, NADP+, FMN
The sulphonamides competitively inhibit and FAD) and biological oxidations
the biosynthesis of folic acid at the step where The redox coenzymes have important func-
p-aminobenzoic acid is incorporated. Higher tions in biological oxidation and reduction
animals cannot synthesize folic acid and reactions. Biochemical oxidations usually take
require it in their diet as a vitamin. Their place by removal of pairs of hydrogen atoms
metabolism is therefore unaffected by the from the molecules to be oxidized. A typical
sulphonamides. Bacteria however synthesize example is the oxidation of malate in the TCA
their own folic acid and growth of these organ- cycle shown in Figure 6.11.
isms is selectively inhibited by sulphonamides. Although the reactions involving these
coenzymes are oxidations they do not in
Allopurinol A further example of a common- themselves require oxygen. However the
ly used enzyme inhibitor is allopurinol. This reduced coenzymes may later be re-oxidized
compound is used in the treatment of gout: a in the electron transport chain, which does
condition which occurs in man and poultry require oxygen.
p-aminobenzoic acid a sulphonamide
o o
".~">
~.)lN N H
"l):)·N N
H
hypoxanthine allopurinol
Figure 6.10 Many pharmaceuticals act as competitive inhibitors of enzymes because they resemble their
substrates. Sulphonamides resemble p-aminobenzoic acid and inhibit bacterial synthesis of folic acid.
Allopurinol is an analogue of hypoxanthine which is used in the treatment of gout.
When the pairs of hydrogen atoms are during which ATP is synthesized. It is there-
removed they are transferred to a hydrogen fore mainly involved in energy conservation.
acceptor such as NAD+, NADP+, FMN or FAD. On the other hand, NADPH is used as a reduc-
NAD+ (nicotinamide adenine dinucleotide) ing agent and serves as a source of hydrogen
and NADP+ (nicotinamide adenine dinu- atoms to be used in biosynthetic reactions.
cleotide phosphate) differ chemically only in NAD+ and NADH act as normal substrates
the fact that NADP+ has an extra phosphate for their enzymes and so are not attached per-
group. Their structures are shown in Figure manently to anyone enzyme molecule, but
6.12. Despite their chemical similarity, the are temporarily bound to the active site during
functions of NADH and NADPH are usually the reaction.
quite different. NADH is almost invariably NADH strongly absorbs UV light at a wave-
reoxidized in the electron transport chain, length of 340 nm whereas NAD+ absorbs only
C02H
6H 2
+NAD
+
+ NADH+ H+
H-6-0H
60 H 2
Malate oxaloacetate
Figure 6.11 A typical redox reaction involving NAD+/NADH. NAD+ accepts a pair of hydrogen atoms
from malate as it is oxidized to oxaloacetate.
86 Enzymes
NH2
I
N ?' N" " ONH 2
~ / 0 0
6- 6-
o 0 0 0
H H H H
2 electrons +
2H
aON~
N
I
Oxidised form (NAD+) Reduced form (NADH)
R R H
CH3(X~yNy CH3(X~X;~Y
CH3 N~NH CH3 N NH
o A 0
FAD
Figure 6.12 NAD+/NADH and FAD/FADH z are important redox coenzymes. Each may be converted from
oxidized to reduced form by accepting hydrogen atoms from a substrate which is oxidized.
weakly at this wavelength. The formation or it is possible to couple reactions which do not
disappearance of NADH can therefore be fol- involve these coenzymes to others which do,
lowed continuously in a spectrophotometer by so that they can also be followed in this way.
measuring the absorbance at this wavelength. Like NAD+, the flavin coenzymes (FAD and
As many enzymes use either NAD+ or NADH FMN) also act as carriers of hydrogen atoms
as substrates, it is relatively easy to make kinet- during oxidation and reduction reactions but
ic measurements of their reactions. In addition, their actions differ in several ways:
• the flavin coenzyme is attached firmly to aGO, the standard free energy change. A solu-
the enzyme molecule, and for this reason it tion with aIM hydrogen ion concentration
is a prosthetic group; has a pH of zero, but in biochemistry it is more
• the flavin can accept either one hydrogen usual to discuss energy changes taking place
atom (to form FAOH or FMNH) or two at pH 7, as these approximate more closely to
hydrogen atoms (to form FAOH 2 or conditions in the cell. The standard free ener-
FMNH2) - Figure 6.12. gy change taking place under these conditions
is called aGO' and it is related to the equilibri-
Subsequently FAOH 2 can be converted back um constant for a reaction by the equation:
to FAD when the hydrogen atoms are passed
to the electron transport chain. aGO' = -RTlnKeq' Equation 6.8
where R is the gas constant and T is the
absolute temperature.
ATP and ADP
Values of aGO' calculated from this equation
Adenosine triphosphate (ATP) and adenosine for different values of K' eq are given in Table 6.3.
diphosphate (AOP) are also two very important Thus irreversible reactions have large nega-
coenzymes. They are not vitamins, however, as tive values of aGO' (This is a rather simplified
they can be synthesized by animals and plants. view of energetics. An irreversible reaction
Many of the reactions in biochemical pathways actually has a large negative value for aG, the
(particularly those involving sugars) take place free energy change accompanying the reac-
using substrates that are linked to phosphate tion under the actual conditions in the cell, not
groups, and it is mainly from the terminal ()') aGo', which assumes that all reactants and
phosphate group of ATP that these are derived products are present at 1 M concentrations.
(Figure 6.13). In addition the conversion of ATP However comparison of aGo' values is suffi-
into AOP is accompanied by the release of con- ciently rigorous for the purposes of this text.)
siderable amounts of energy which can be used One very useful characteristic of phosphate
to drive reactions. esters and other phosphate compounds is
All reactions reach an equilibrium: their free energy of hydrolysis, i.e. the free
energy change which accompanies hydrolysis
aA + bB ~ cC + dO Equation 6.6
of a mole of the compound. Some typical val-
The equilibrium constant (Keq) is given by: ues are given in Table 6.4. These compounds
can be roughly divided into two groups:
Equation 6.7 • those with a normal free energy of hydroly-
sis between 0 and -20 kJ mol-1
• high energy compounds, i.e. aGo', more
Where A and B are reactants, C and 0 the
negative than -20 kJ mol-1(ATP and above
products. Some reactions however are essen-
in Table 6.4).
tially irreversible and, for all practical purposes,
take place in one direction only. In these cases, Also aGo' values are additive.
at equilibrium, the concentrations of the prod- An especially useful feature of Table 6.4 is
ucts [C] and [0] are much greater than those of that the energy released during a reaction
the reactants [A] and [B]. Thus, Keq is very large. high up the table can be used to drive the
The free energy of a reaction is the energy reversal of reactions lower down. Thus a phos-
difference between products and reactants. phate group can effectively be transferred
When this is measured under standard condi- from the high-energy compounds to other
tions of concentration (1 M), temperature (25 0
molecules, to produce lower-energy phos-
C) and pressure (1 atm) this is referred to as phate esters. These points can be illustrated by
88 Enzymes
NH2
I
N~N
~ .. ) - ) 0 0 0
N ~0'jCH,oJ~~
);----f,'" 6 6- 6
H H ATP
NH2
I
N N
l,,) 0 0
6- 6-
ADP
H H
Inorganic phosphate (Pi)
Figure 6.13 The structure of ATP and its breakdown products. Hydrolysis of ATP normally produces
ADP and inorganic phosphate (P). The reaction releases a considerable amount of energy because of the
relative instability of ATP and stability of Pj'
the first step in glycolysis, the conversion of However this reaction is unfavourable and
glucose into glucose-6-phosphate, which will would not occur spontaneously because of the
be discussed in more detail in Chapter 10. size of the free energy change. At equilibrium
This reaction might occur by simple con- the reaction mixture would contain mainly
densation of glucose with a molecule of inor- glucose and Pi (Keq -0.01).
ganic phosphate (P;) as follows: An alternative reaction for the production of
glucose-6-phosphate from glucose is as follows:
glucose + Pi ~ glucose-6-phosphate + H 20
dGD' = + 13_8 kJ mol-1 glucose + ATP ~ glucose-6-phosphate + ADP
Table 6.3 Free energy changes and equilibrium constants
K' eq ~GO'
(kJ mol-I) (kcal mol-I)
0.00001 28.5 6,82

0.001 17,1 4.09
0.1 5.7 1.36
1.0 0.0 0.00
10.0 -5.7 -1.36
1000.0 -17.1 -4.09
100000,0 -28.5 -6.82 - IRREVERSIBLE
Table 6.4 Free energy of hydrolysis of phosphate esters
Reaction LlCD'
(kJ moP)
Phosphoenolpyruvate + H 20 -> pyruvate + Pi -61.9

1,3 Diphosphoglycerate + HzO -> 3 phosphoglycerate + Pi -49.3
Creatine phosphate + H 20 -> creatine + Pi -43.1
Acetyl phosphate + H 20 -> acetate + Pi -42.2
ATP + HzO -> ADP + Pi -30.5
Glucose-I-phosphate + H 20 -> glucose + Pi -20,9
Glucose-6-phosphate + H 20 -> glucose + Pi -13.8
Glycerol-I-phosphate + H 20 -> glycerol + Pi -9.2
This reaction can be considered in two parts energetically favourable process of simple
which can be summed to obtain the overall hydrolysis as follows:
equation:
glucose-6-phosphate + H 20 ~ glucose + Pi
glucose + Pi ~ glucose-6-phosphate + H 20 LlGo' = -13.8 kJ mol-1 Equation 6.10
LlGo' = + 13.8 kJ mol-1
This example illustrates the very general
point that reactions in pathways for the biosyn-
ATP + Hp ~ ADP + Pi
thesis and breakdown of biochemical com-
LlGO' = -30.5 kJ mol-1
pounds usually differ from one another at key
points, as this allows the energetics of both
glucose + ATP ~ glucose-6-phosphate + ADP
steps to be favourable. It also allows indepen-
LlGo' = -16.7 kJ mol-1 Equation 6.9
dent control of the rate of both pathways. The
For this reaction Keq -1000 so the equilibri- continuous synthesis of glucose-6-phosphate
um position favours production of glucose-6- from glucose and its subsequent hydrolysis back
phosphate, which takes place readily. A conse- to glucose by the reactions described above rep-
quence of this, however, is that the reverse resents a 'futile cycle' in which ATP is wasteful-
reaction is extremely unfavourable. Hence in ly hydrolysed. This can be prevented in the cell
gluconeogenesis (see Chapter 18), glucose-6- by ensuring that only glucose-6-phosphate syn-
phosphate cannot be converted into glucose thesis or its breakdown takes place rapidly at
by transferring its phosphate group to ADP. one time, which is achieved by regulating the
This transformation is easily achieved by the activity of enzymes catalysing the reactions.
90 Enzymes
By far the commonest compound which compound. This is called orthophosphate
acts as the donor of phosphate groups, and cleavage, but in some cases the two terminal
which therefore drives reactions in this way, is phosphates are removed together in the
ATP. The vital role of this compound in phos- process of pyrophosphate cleavage.
phate group transfer, and in providing the
chemical energy needed to make otherwise
ATP ~ AMP + PPi .:lco' = -41.8 kJ mol-I
energetically unfavourable reactions take where AMP = adenosine monophosphate
place, is determined by two main factors. and PPi = pyrophosphate. This can provide
more push for the reaction because .:lOY is
• The free energy of hydrolysis of ATP (-30.5
higher and because tissues contain pyrophos-
kJ mol-I) is high, so that when the conver-
phatases which catalyse the hydrolysis of
sion of ATP into ADP occurs, sufficient
pyrophosphate to two molecules of inorganic
energy is made available to drive other
phosphate. This irreversible process removes
reactions by ensuring that the overall .:lco'
the product of the first reaction and hence
value is negative.
pulls the equilibrium further to the right.
• The free energy of hydrolysis of ATP is not
Reactions in which pyrophosphate cleav-
excessive. More energy could be provided
age of ATP occurs include:
to drive reactions by one of the compounds
higher up Table 6.4, but towards the top of • activation of fatty acids, which precedes 13-
the list the compounds become more and oxidation
more difficult to make because they require RCOzH + ATP + CoASH -+ AMP + PPi +
a great deal of energy for their synthesis. R-CO-SCoA
• formation of amino acyl tRNAs in protein
The particular value of ATP therefore is that
synthesis
it is near the middle of the table and, although
amino acid + tRNA + ATP -+ aminoacyl
a high-energy compound, is relatively easily
tRNA + AMP + PPi
made by linking its synthesis to other reactions
• formation of nucleoside diphosphate sug-
releasing even more energy, such as conversion
ars in di- and polysaccharide synthesis
of l,3-diphosphoglycerate to 3-phosphoglycer-
ATP + G-1-P -+ ADP-glucose + PPi
ate, hydrolysis of phosphenol pyruvate, or
processes occurring during electron transport.
6.6 ALLOSTERIC ENZYMES
The free energy of hydrolysis is the differ-
ence in energy between the starting com- The enzymes discussed so far are simple
pounds (ATP) and the products (ADP + PJ In enzymes which obey the Michaelis-Menten
the case of ATP, hydrolysis yields a consider- kinetic equations. The derivation of these
able amount of energy (Figure 6.13) for the fol- equations assumes that each enzyme mole-
lowing reasons. cule behaves independently of others.
However some enzymes, known as allosteric
• ATP is unstable as a result of the four nega- enzymes, consist of a number of subunits,
tive charges which it carries at normal pH,
each composed of one or more polypeptide
which repel one another. This repulsion is
chains. The subunits are often identical in
much reduced in ADP because there are
structure but may be different. These sub-
fewer charges.
units carry the active site, to which the sub-
• Phosphate is especially stable because of the strate binds, and one or more regulatory sites
existence of resonance structures.
which bind signal molecules (effectors). The
In most reactions in which ATP is involved, binding of substrate to an active site or the
the terminal (-y) phosphate is transferred binding of an effector to a regulatory site
either to water (hydrolysis) or to some other induces a conformational change in the sub-
Molecular recognition 91
unit which may influence other active sites 6.7 MOLECULAR RECOGNITION
and either increase or decrease their affinity
One of the most characteristic and important
for the substrate. These effects can be trans-
properties of proteins is their ability to bind or
mitted from one subunit to another so that
'recognise' other molecules. The action and
the active site and regulatory sites may be on
specificity of enzymes depends on this proper-
different subunits. Where the binding of sub-
ty, but many other proteins which are not
strates affects other active sites the kinetics
enzymes, such as receptors and antibodies,
are usually complex, and often a plot of va
also show specific binding.
against [5] will not be a hyperbola but a sig-
moid curve (Figure 6.14). This interaction
between subunits is called co-operativity. 6.7.1 RECEPTORS
Where binding of an effector affects the A receptor is a molecule which binds, and
active site, complex patterns of either inhibi- thereby detects the presence of, another mole-
tion or activation may be seen as a result of cule. Thus for example the binding of a hor-
positive or negative co-operativity. Inhibition mone to its receptor may initiate a response
of the activity of allosteric enzymes by the which will result in the changes which are
end-product of metabolic pathways is partic- characteristic of that hormone. Binding to
ularly important in the control of many meta- receptors is analogous to binding of substrates
bolic pathways (see Chapter 22). to enzymes and displays high affinity and
Models developed to describe the mecha- specificity. The nature of receptors for some
nism of allosteric enzymes propose that the hormones is discussed further in Chapter 20.
enzyme subunits exist in two forms with
either low or high affinity for the substrate,
6.7.2 ANTIBODIES
and that they may be switched between these
forms by binding of substrate or effector Antibodies are proteins which animals pro-
(Figure 6.15). duce in response to materials (antigens), usu-
100
90
80
70
S 60
e
50
~III
40
~
30
20
10
0
0 20 40 60 80 100
S
Figure 6.14 Kinetics of an allosteric enzyme. Allosteric enzymes often show sigmOid kinetics because of
cooperation between the subunits of which they are composed.
92 Enzymes
®®
®®
Figure 6.15 Structure of an allosteric enzyme showing its conversion from a low- to a high-activity form
by binding an effector (E). Binding of E to a regulatory site causes a conformation change, increasing the
affinity of the catalytic site for substrate (S).
ally from outside the body. Antibodies form arms are held together by a single bridge. The
part of the defence mechanism of the immune part of the molecule which will recognise and
system. They may for example be produced in react with the antigen is contained in the light
response to bacteria or virus coat proteins.
Antibodies form a complex with antigens and
neutralize their effects as a precursor to their
destruction, thus protecting the animal.
Antibodies are extremely specific and can
differentiate not only between proteins from
different species but also between the same
protein from different individuals of the same
species.
Antibodies in general belong to a class of
proteins known as immunoglobulins. Within
this class there are five main groups known as
IgA, IgO, IgE, IgG and IgM. Each of these has
a well-defined role in combating the invasion /
/
of foreign materials. IgG is the antibody which /
s-s
circulates in the blood, whilst IgA acts in the Hinge
region S-S
secretions of the digestive, urinary, respiratory
and reproductive tracts. The discussion in this
section will be limited to IgG. -- _ - - Heavy
__ chains
Each antibody molecule is composed of a

number of individual protein chains which are
classified according to their molecular weight
as heavy or light. Each IgG molecule is made up
of two heavy chains and four light ones (Figure
6.16). The molecule has a characteristic Y shape
with two heavy chains forming the stalk of the
Figure 6.16 The structure of an antibody.
Y upon which are hinged the two pairs of light Antibodies consist of light and heavy chains,
chains that each form the arms. The two heavy linked together through a 'hinge'. The light chains
chains are held together with a pair of disul- contain the part of the molecule which recognises
phide bridges. The light chains in each of the and reacts with the antigen.
Molecular recognition 93
chains. This area is similar in its binding and gen causes a change in the shape of a 'univer-
specificity to the active site of an enzyme. sal' antibody in such a way that it induces
The means by which cells are able to syn- complementarity in the antibody.
thesize a different antibody in response to Recent discoveries concerning the roles of
almost any challenge encountered is a source proteins in the formation of the tertiary struc-
of much current debate. Basically the two ture of other proteins, as is found in prions and
extreme possibilities are either that all cells with the heat-shock proteins, add weight to the
contain the genetic information to produce possible roles of the antigens in determining
each type of antigen protein; or that each anti- the form of their appropriate antibodies.
PURINES, PYRIMIDINES AND NUCLEIC 7
ACIDS
7.1 Introduction 95
7.2 Purines and pyrimidines 95
7.3 Deoxyribonucleic acid (DNA) 96
7.3.1 Chemical nature of DNA 96
7.3.2 The DNA double helix 98
7.3.3 Structure of DNA in prokaryotes and 98
eukaryotes
7.3.4 Organelle DNA 100
7.4 Ribonucleic acid (RNA) 100
7.4.1 Messenger RNA (mRNA) 101
7.4.2 Transfer RNA (tRNA) 101
7.4.3 Ribosomal RNA (rRNA) 102
7.1 INTRODUCTION the sugar is ribose and in DNA it is deoxyri-

The properties of all proteins are determined bose. Each of the sugars carries a purine or
by the sequence of the 20 different amino acids pyrimidine base, and it is the sequence in
which they contain. Any errors in these which these bases occur that provides the
sequences usually prevent them from per- information needed to code for the amino
forming their normal biological functions. The acids in proteins.
order in which the amino acids are assembled
is determined by the structure of the DNA
7.2 PURINES AND PYRIMIDINES
(deoxyribonucleic acid) molecules in the cells.
DNA therefore acts as the 'blueprint' which Purines and pyrimidines are small, nitrogen-
determines the nature and function of pro- containing, organic bases. Pyrimidines consist
teins. DNA also has the vital function of trans- of a single ring whilst purines contain two
mitting this information from one generation fused rings. The purine and pyrimidine bases
to the next, as the information in the DNA can are rarely found as free compounds; they are
be copied and handed on to daughter cells. normally attached to sugar groups to form
DNA is a type of nucleic acid. Although the nucleosides, or to sugar phosphates (as in the
information required for the synthesis of pro- nucleic acids) to form nucleotides (Table 7.1).
teins is carried in DNA, another type of nucle- The structures of the purine and pyrimidine
ic acid called RNA (ribonucleic acid) is also bases which are found in most RNA and DNA
required for the translation of the information are shown in Figure 7.1. In addition a number
in the DNA into the sequence of amino acids of unusual bases are found in some specific
in a protein. types of RNA. As well as their role in nucleic
Both DNA and RNA consist of a backbone acids, some purine and pyrimidine derivatives
of alternating sugar and phosphate groups (e.g. ATP, NADH) are important coenzymes
forming very long chains. In the case of RNA and are involved in energy metabolism.
96 Purines, pyrimidines and nucleic acids
Cytosine Uracil Thymine
Pyrimidine bases
NH2
())
I
N N
H
Adenine Guanine
Purine bases
Figure 7.1 Structure of the purine and pyrimidine bases which are commonly found in RNA and DNA.
Pyrimidine bases: cytosine, uracil, thymine; purine bases: adenine, guanine.
7.3 DEOXYRIBONUCLEIC ACID (DNA) The base may be either a purine or a pyrimi-
7.3.1 CHEMICAL NATURE OF DNA
dine. The purines are adenine (A) or guanine
(G), and the pyrimidines cytosine (C) or
The basic structure of DNA is shown in Figure thymine (T). DNA is mostly double stranded.
7.2. The sugar in DNA is deoxyribose. Each The two strands run alongside but in opposite
sugar carries a base attached to the I' position. directions and are described as antiparallel.
Table 7.1 Some purine and pyrimidine bases, nucleosides and nucleotides
Base Nucleoside Nucleotide
Adenine Adenosine (A) AMP (Adenosine monophosphate)

Guanine Guanosine (G) GMP (Guanosine monophosphate)
Hypoxanthine Inosine (I) IMP (Inosine monophosphate)
Uracil Uridine (U) UMP (Uridine monophosphate)
Thymine Thymidine (T) TMP (Thymidine monophosphate)
Cytosine Cytidine (C) CMP (Cytidine monophosphate)
Deoxyribonucleic acid (DNA) 97
3'end
S'end I
H o
I
'O-p=O
I
o
I CH2
5' CH2 I
4'
o
I
'O-P=O
I
H",
o H H 0
I
'0- P=O
I
o
I
CH 2
r----,. ::::::1 BASE
o
I
'O-P=O
I
o H H 0
I
'0- P=O
I
o
I CH 2
CH2 I
o
I
'O-P=O
I
R",
o H H 0
I
'0- P=O
I
o
I
CH2
r----, J IlASE
o
I
'O-P=O
o H I
o
3'end S'end
Figure 7.2 Diagrammatic representation of the structure of DNA. Each chain consists of a backbone of
alternating deoxyribose sugar and phosphate groups. The sugars carry a purine or pyrimidine base
which forms hydrogen bonds with complementary bases in the second chain. The two chains run in
opposite directions and are therefore antiparallel.
The two chains are held together by hydro- thymine or between cytosine and guanine
gen bonds formed between adenine and (Figure 7.3).
7.3.2 THE DNA DOUBLE HELIX
Auo . . . . H-~-H
CH 3
The structure of DNA forms a double helix in
r' r ~N which the two sugar phosphate chains make
/N'(N-Hl.N up the backbone of the molecule and are twist-

ed around one another in a spiral. The bases,
which are paired together, point inwards
towards one another and lie perpendicular to
I the axis of the helix. They thus resemble the
Thymine Adenine rungs on a spiral ladder. One turn of the helix
occurs roughly every ten nucleotides and
occupies about 3.4 nm of the length of the
chain. Two grooves, the narrow groove an~
the wide groove, can be seen along the surface
H
of the helix and these provide possible posi-
I
( I N - H ........ 0 tions for interaction of the DNA with other
molecules such as proteins (Figure 7.4). The
;' Ny~ H_N~)N, DNA helix itself may undergo a type of coiling
O········H-N~~
called supercoiling.
I N
H I 7.3.3 STRUCTURE OF DNA IN PROKARYOTES
AND EUKARYOTES
Cytosine Guanine There are differences in the organisation of
Figure 7.3 Hydrogen bonding between pairs of DNA in prokaryotes and eukaryotes. In bacte-
bases in DNA. The bonding is very specific: two ria such as Escherichia coli the DNA is circular
bonds form between adenine and thymine and and found in the nucleoid region of the cyto-
three between cytosine and guanine. In each pair plasm, where it is associated with small
one base is a purine and one a pyrimidine. amounts of proteins and folded to form about
100 supercoiled loops. In eukaryotes DNA
This hydrogen bonding (base pairing) is exists in the form of well-defined chromo-
extremely specific, always involving these somes within the nucleus. These chromo-
particular combinations of purine-pyrimi- somes are made up of chromatin in which
dine pairs. This means that in double-strand- DNA forms a complex with large quantities of
ed DNA there are always roughly equal basic proteins including histones. The compo-
quantities of purines and pyrimidines. Three sition of chromatin is given in Table 7.2.
hydrogen bonds can be formed between Histones are small, basic proteins of molec-
cytosine and guanine but only two between ular weight about 10-20 kDa containing a
adenine and thymine. DNA containing a large proportion of basic amino acids such as
high proportion of C and G is more stable to arginine and lysine. They have large numbers
heat than that containing a lower proportion. of positive charges and readily form complex-
The formation of these base pairs can only es with negatively charged nucleic acids.
occur when the chains are arranged in an Lysine residues in the histones may be acety-
antiparallel formation. The two chains of the lated or methylated, and serines may be phos-
DNA are said to be complementary, as the phorylated. Each of these changes reduces the
bases of one are determined by, and are com- net positive charge and may reduce binding to
plementary to, the other. DNA.
Deoxyribonucleic acid (DNA) 99
... Narrow groove
p.4 om I
... Wide groove
Figure 7.4 The DNA double helix. The two chains are held together by hydrogen bonds between their
complementary bases and are twisted into a helix. There are approximately 10 nucleotides to each turn
which occupies about 3.4 nm along the length of the chain. A wide and a narrow groove run along the
surface of the helix.
The non-histone proteins may playa role in within the chromosomes. In a nucleosome,
switching on and off genes in some parts of DNA is wound around eight histone subunits
the chromatin. They may do this for example and these are themselves coiled around a hol-
in response to hormones which activate spe- low core so that in the chromosome the DNA
cific genes. forms part of a coiled coil (Figure 7.5).
Through the electron microscope, repeating Nucleosomes themselves may occur in rela-
structures called nucleosomes can be identified tively open or closely packed arrangements
Table 7.2 The composition of chromatin
Component Relative amount

(DNA =100)
DNA 100
Histones 114
Non-histone proteins 33
RNA 7
but in this case the sugar is ribose not deoxyri-
Histone
bose. There is an OH group at the 2' position
of the ribose (Figure 7.6).
The bases also differ slightly from those
found in DNA. In particular, thymine is not
Linker DNA found in RNA, but the pyrimidine uracil
I
·0- P=O
I
o
I
5' CH2
4'
v 0 BASE
l'
3'
o OH
I
·0- P=O
I
o
I
Figure 7.5 Nucleosome structure. In eukaryotes,
6H I
Q
nucleosomes are observed at at intervals along the 0 BASE
DNA. Each nucleosome consists of approximately
140 base pairs of DNA wrapped around eight his-
tone subunits. The region between nucleosomes is
called linker DNA and is between 20 and 100 base
pairs long, depending on the species. Nucleosomes o OH
may themselves adopt a helical arrangement to I
·0- P=O
form fibres or solenoids.
I
o
o
and it is believed that in the latter genes can-
not be expressed, whilst in the former they tH2 0 I BASE I
may be activated under some circumstances.
7.3.4 ORGANELLE DNA

o OH
In addition to the DNA found in the nucleus, I
·0- P=O
small quantities are also found in chloroplasts
and mitochondria. The information carried in
I
o
this DNA codes for some of the proteins of I
CH 2 0 BASE
these organelles. This DNA resembles
prokaryotic DNA. Organelle genomes are dis-
cussed in more detail in Chapter 21.
7.4 RIBONUCLEIC ACID (RNA) o OH

Figure 7.6 Diagrammatic representation of the
Chemically RNA is very similar to DNA but structure of RNA. The backbone consists of alter-
there are some important differences. nating ribose sugars and phosphate groups. Each
Like DNA, it consists of a backbone made sugar carries either a purine or pyrimidine base.
up of alternating sugar and phosphate groups, RNA is normally single-stranded.
Ribonucleic acid (RNA) 101
occurs instead. The bases found in most RNA are several thousand types of mRNA in most
molecules are thus adenine, guanine, cytosine cells. In eukaryotes one type of mRNA mole-
and uracil. cule corresponds to each type of protein, but
Most RNA is single-stranded, but within its in prokaryotes some mRNA is polycistronic. In
structure short regions may be coiled to form this case a mRNA molecule may carry the code
base-paired regions. for several different proteins. The molecular
There are three distinct types of RNA: weight of mRNA is thus very variable, particu-
larly in prokaryotes.
• messenger RNA (mRNA)
In prokaryotic cells mRNA is made on the
• transfer RNA (tRNA)
DNA within the nucleoid region of the cyto-
• ribosomal RNA (rRNA).
plasm and is then used immediately to code
These differ from one another in their size for protein synthesis. In eukaryotes, however,
and function as will be seen below, but chem- most mRNA is made in the nucleus, from
ically they are very similar. The synthesis of all where it passes out through the nuclear enve-
three types is catalysed by enzymes called lope to the cytoplasm. Before transfer to the
RNA polymerases. Unlike DNA synthesis, the cytoplasm introns may be removed and 3' or 5'
formation of RNA is not restricted to times of caps may be added (see Chapter 21).
cell division but takes place throughout the life In prokaryotes, mRNA molecules turn over
of the cell. very rapidly. This means that each one is read
The sequence in which bases are assembled by a ribosome only a few times before it is
into RNA is determined by the order of bases destroyed. This allows the cell to respond very
in DNA. Before RNA synthesis can occur the rapidly to the changing environment to which
strands of DNA must separate from one it is exposed. On average each mRNA molecule
another. RNA is synthesized on the template is broken down after only 2-3 minutes. In
strand of DNA which is therefore complemen- eukaryotes the stability of different mRNA
tary to this strand. The other strand has the molecules varies, but on the whole they are
same sequence of bases as the RNA produced, much more stable than in prokaryotes. The
except that T replaces U and thus is called the average half-life of eukaryotic mRNA is about
coding strand. The bases in the template 10 hours but some types persist much longer
strand of DNA and the complementary ones than this. In reticulocytes, for example, haemo-
inserted into RNA are noted in Table 7.3. globin continues to be synthesized even after
the nucleus disappears because these cells con-
tain stable mRNA which codes for this protein.
7.4.1 MESSENGER RNA (mRNA)
Some mRNA is synthesized in the chloro-
mRNA makes up about 2% of the total RNA. It plasts and the mitochondria as part of the
contains the sequence of bases needed to process of protein synthesis occurring in these
determine the order in which amino acids will organelles.
be assembled into each type of protein. There
Table 7.3 Relationship between bases in DNA 7.4.2 TRANSFER RNA (tRNA)
and RNA
tRNA makes up about 16% of the total RNA.
Template strand of DNA Complementary RNA tRNA molecules are small and uniform in size,
having molecular weights in the range of
Adenine Uracil 23-30 kDa. This corresponds to between 75
Thymine Adenine and 90 nucleotides.
Cytosine Guanine The system for coupling amino acids
Guanine Cytosine
together to form proteins cannot recognise
the different amino acids directly. Instead it ribosomes, the organelles on which protein
uses tRNA as an intermediate carrier. Each synthesis takes place.
molecule of tRNA has a site for attachment of Ribosomes consist of two subunits: a large
amino acids and another which is comple- one and a small one. They contain about 65%
mentary to, and recognises, sections of rRNA and 35% protein. The ribosomes in
mRNA molecules. tRNA thus brings amino prokaryotes differ in size from those in
acids to the ribosomes where they are assem- eukaryotes. It is convenient to use their sedi-
bled into proteins. There is at least one type mentation constant, which is measured in
of tRNA molecule for each of the 20 amino Svedberg units (5), as a measure of their size.
acids which occur in proteins, and for some This measures how rapidly they sediment in a
amino acids there are several. There are about centrifuge when subjected to a particular grav-
50 different types of tRNA in anyone cell, at itational force. Large particles have large 5 val-
least one type for each amino acid found in ues. They are not additive, in other words join-
proteins. ing together a 60S particle with a 405 particle
In addition to the four major bases, tRNA in eukaryotic ribosomes produces an 80S
also contains up to 10% of unusual bases such rather than a 1005 particle.
as pseudouridine, ribothymidine or dihy- 70S ribosomes are characteristic of prokary-
drouracil. Although the exact sequence of otes and of the ribosomes found in the mito-
bases varies from one type of tRNA to anoth- chondria and chloroplasts of eukaryotes, whilst
er, each conforms to a common overall struc- 80S ribosomes are found in the cytoplasm of
ture which can be drawn out as a clover leaf. eukaryotic organisms. Whatever their size,
This has several important parts (Figure 7.7): ribosomes consist of several pieces of RNA and
a number of proteins. The composition of 70S
• an anticodon, consisting of three bases, and 80S ribosomes is described in Figure 7.8.
which can base pair with complementary The differences between 70S and 80S ribo-
bases in mRNA; somes are important because many antibiotics
• an amino acid attachment site, to which a inhibit bacterial protein synthesis on 70S ribo-
specific amino acid becomes attached somes without affecting that taking place on
(Chapter 21); 80S ribosomes. They thus selectively kill bacte-
• other arms which may help binding to the ria and other prokaryotes. More information
ribosomes; the number of nucleotides in all about the effects of antibiotics on protein syn-
of the arms except the variable one is fixed thesis can be found in Chapter 30.
(Figure 7.7). rRNA is made in a specialized region of the
nucleus called the nucleolus. The rRNA for
one ribosome is made in one long piece. It
7.4.3 RIBOSOMAL RNA (rRNA)
then complexes with proteins before being
rRNA makes up more than 80% of the total split into several smaller pieces which make
RNA in the cell. It is a major component of the up the ribosome.
Ribonucleic acid (RNA) 103
3'
Amino acid
OH
attachment site
5'
P
Tille loop
DHU loop
Variable arm
Anticodon
Figure 7.7 The clover-leaf structure of yeast alanine tRNA. Other tRNA molecules also adopt the clover-
leaf structure. The regions which are common to all tRNAs are indicated. The amino acid becomes
attached to the 3' -OH group of the terminal adenosine residue, and the sequence of bases in the anti-
codon is unique to each type of tRNA.
rRNA
/
(55+5.85+ 185+235)
Large subunit
605
Eukaryotic
/ ~I >50 proteins
Ribosome
(805)
/1
rRNA
~
(165)
5mall subunit
405
~I >30 proteins
"'--:---""""-7""""1 /
I
' - -_ _
rRNA
{5_5_+2_3_5_)_--'
Large subunit / -
__
505
.---::---:---~/
Prokaryotic
~ 1~ >__
30__ P_ro_~_i_ns__~
Ribosome
(705)
~ ,--:;::----,""""':---7""""1 / 1
....__r_R_NA_(1_6_5)_--,
5mall subunit / -
__
305
~ 1~ >__
20__
p_ro_te_i_ns__ ~
Figure 7.8 Composition of 80S ribosomes which are found in the cytoplasm of eukaryotes and 70S ribo-
somes found in prokaryotes and eukaryotic organelles. Both types consist of two subunits which contain
both RNA and proteins, but 80S ribosomes are generally more complex.
VITAMINS AND MINERALS 8
8.1 Vitamins in biochemistry 105
Introduction
8.1.1 105
Thiamin (vitamin B1)
8.1.2 106
Riboflavin (vitamin B2)
8.1.3 108
Nicotinic acid (niacin, formerly called
8.1.4 108
vitamin B5)
8.1.5 Pantothenic acid 112
8.1.6 Pyridoxine, pyridoxal and 112
pyridoxamine (vitamin B6)
8.1.7 Biotin 114
8.1.8 Folic acid 114
8.1.9 Vitamin B12 115
8.1.10 Vitamin C 117
8.1.11 Choline 118
8.1.12 Carnitine 119
8.1.13 Vitamin A 119
8.1.14 Vitamin 0 121
8.1.15 Vitamin E 124
8.1.16 Vitamin K 126
8.2 Minerals in biochemistry 127
8.2.2 Calcium 127
8.2.4 Magnesium 128
8.2.5 Sodium, chloride and potassium 129
8.2.6 Sulphur and iron 129
8.2.7 Other elements with known 130
biochemical functions
8.1 VITAMINS IN BIOCHEMISTRY they be identified simply by letters of the

alphabet. This system worked satisfactorily for
8.1.1 INTRODUCTION
a number of years and eventually nine vita-
A detailed knowledge of the structure and the mins were discovered: A, B, C, 0, E, F, G, H
biochemical and physiological roles of vita- and I. However, it was soon discovered that F
mins has been accumulated over the past 50 was not a true vitamin and that vitamin B was,
years. However, before this time diseases were in fact, a mixture of several vitamins. As a
recognised which were related to diet. We result the lettering system, though still in use,
now know that many of these diseases were is now disjointed and a number of vitamins
caused by vitamin deficiencies. are referred to by their chemical names. In
In 1920, before the chemical composition of addition, the vitamins can be classified into
vitamins was known, it was proposed that two groups according to their solubility prop-
106 Vitamins and minerals
erties. Thus the vitamins of the B complex and circumstances an increase in intake may be
vitamin C are known as the water-soluble vit- beneficial. There is little evidence of toxicity
amins, while the remainder are known as the associated with excessive intake of water-sol-
fat-soluble vitamins (Table 8.1). uble vitamins.
In general, fat-soluble vitamins contain only In contrast, fat-soluble vitamins are stored
carbon, hydrogen and oxygen, whereas the in body tissues. As their name suggests they
water-soluble vitamins may also contain vary- are found in the fat depots of the body, which
ing proportions of nitrogen, sulphur or cobalt. may act as a reserve for times of inadequate
Fat-soluble vitamins can occur in plant tissue intake. The liver is also rich in fat-soluble vita-
in the form of provitamins: vitamin precursors mins. Because the body has the ability to build
which can be converted into vitamins in the up reserves of these vitamins it is possible for
animal body. No provitamins are known for them to accumulate to toxic concentrations.
the water-soluble vitamins.
The body has little capacity to store water- 8.1.2 THIAMIN (VITAMIN Bj )
soluble vitamins and animals require a regu-
Biochemical functions
lar supply of small quantities of them.
Amounts consumed in excess of require- In cells thiamin occurs largely as its active
ments are excreted, usually via the urine. coenzyme form, thiamin pyrophosphate (for-
Thus, supranormal intakes of water-soluble merly cocarboxylase) (Figure 8.1). It acts as a
vitamins have little effect on long-term body coenzyme for two types of enzyme-catalysed
reserves. The body's requirement for vita- reactions involved in carbohydrate metabo-
mins changes with age, physiological status lism. The first is the decarboxylation of a-keto
and during illness and infection. Under these acids. This type of reaction is important in the
Table 8.1 Vitamins, their coenzyme forms and main functions
Type Coenzyme or active form Main Function
Water-soluble
Thiamine (B j ) Thiamine pyrophosphate (TPP) Aldehyde group transfer
Riboflavin (B 2) Flavin mononucleotide (FMN) Hydrogen atom (electron) transfer
Flavin adenine dinucleotide (FAD) Hydrogen atom (electron) transfer
Nicotinic acid Nicotinamide adenine dinucleotide Hydrogen atom (electron) transfer
(NAD)
Nicotinamide adenine dinucleotide Hydrogen atom (electron) transfer
phosphate (NADP)
Pantothenic acid Coenzyme A (CoA), acyl carrier protein Acyl group transfer
(ACP)
Pyridoxine (B6) Pyridoxal phosphate Amino group transfer
Biotin Biocytin Carboxyl group transfer
Folic acid Tetrahydrofolic acid One-carbon group transfer
Cyanocobalamin (B I2 ) Coenzyme BI2 1,2 shift of hydrogen atoms
Ascorbic acid (C) Coenzyme in hydroxylation
Fat-soluble
Vitamin A ll-cis-Retinal Visual cycle, bone growth, cell
differentiation
Vitamin 0 1,25-Dihydroxycholecalciferol Calcium and phosphate metabolism
Vitamin E a-tocopherol Antioxidant
Vitamin K Prothrombin biosynthesis
Vitamins in biochemistry 107
catabolism of sugars and the TCA cycle Xylulose-5-P + ribose-5-P -->
(Chapter 11) and examples are:- sedoheptulose-7-P + glyceraldehyde-3-P
• The conversion of pyruvate to acetyl-CoA
catalysed by pyruvate dehydrogenase.
Metabolism of thiamin
Pyruvate + NAD+ + CoA -->
The metabolism of thiamin can be inhibited by
acetyl-CoA + NADH + H + + CO 2
the presence of synthetic analogues (Figure
• The conversion of a-ketoglutarate into suc- 8.1). Pyrithiamin blocks the phosphorylation
cinyl-CoA catalysed by the enzyme 0'- of thiamin preventing the formation of the
ketoglutarate dehydrogenase. active coenzyme. Oxythiamin competes with
thiamin pyrophosphate for binding sites on
a-ketoglutarate + NAD+ + CoA -->
enzymes. Amprolium is an anticoccidial agent
succinyl-CoA + NADH + H+ + CO 2
used in the poultry industry. Its effect is to dis-
The second type of reaction involving thi- rupt thiamin metabolism in coccidia but, if
amin pyrophosphate occurs in the pentose given in high concentration, it can affect the
phosphate pathway and is catalysed by trans- host animal by inhibiting thiamin absorption
ketolase enzymes (Chapter 15). and blocking phosphorylation.
N
H,C""""""CXNH2 CH 0- O-
,
N
H C""'v:"CX
II"NH2 CH
, II " , I I_
N /. J-.:.-CH;CHzOH
N /. ~H;CHzO-P-o-P-o
H-N~,
2 \Ls
I CH-N
z \Ls
II
0
II
0
Thiamin
Thiamin pyrophosphate (TPP)
N
N
H,C""'v:"CX
II /.
"
OH CH
CH;-N ~
b I.
CH-CH-QH
2 Z
Pyrlthlamln
Oxythlamln
H,C-CH;CH Z
ycxN
Br- HN+ /.
NH
CH-N
2
2CH,
0'\
_
Br-
Amprollum
Figure 8.1 The structures of thiamin, the coenzyme thiamin pyrophosphate (TPP), and the thiamin ana-
logues oxythiamin, pyrithiamin and amprolium.
Thiaminases are found in a number of 8.1.4 NICOTINIC ACID (NIACIN, FORMERLY
microorganisms and plants, and in certain CALLED VITAMIN B5)
types of raw fish. They split thiamin into its
component pyrimidine and thiazole moieties
and thus destroy its activity. Non-enzymic The physiologically active form of nicotinic
destruction of thiamin may also result from acid is nicotinamide (Figure 8.3) and as such
the presence of reactive substances such as it is an important component of two coen-
caffeic acid, tannins, rutin and quercitin in zymes, NAD+ and NADP+. These coenzymes
feedstuffs. are essential for enzyme-catalysed reactions
The biochemical bases of the symptoms of in carbohydrate, protein and lipid metabo-
thiamin deficiency are not clearly understood. lism. They function in oxidation-reduction
A summary of the deficiency symptoms in dif- reactions by acting as hydrogen transfer
ferent species is given in Table 8.2. agents (Chapter 6). Examples of reactions
involving NAD+ and NADP+ are listed
below:
8.1.~ RIBOFLAVIN (VITAMIN B2)
• interconversion of pyruvate and lactate
Biochemical functions CH3COCOOH + NADH + H+->
CH3CHOHCOOH + NAD+
Riboflavin is a component of flavin mononu-
cleotide (FMN) and flavin adenine dinucleotide • oxidation of 3-hydroxyacyl-CoAs in fatty
(FAD) (Figure 8.2), which are coenzymes acid oxidation
involved in oxidation/reduction reactions
RCH2CHOHCH2COSCoA + NAD+ ->
(Chapter 6). Numerous enzymes containing RCH2COCH2COSCoA + NADH + H+
FMN and FAD are found in all living organisms
and are usually referred to as flavoproteins. • deamination of amino acids
Examples are listed below: amino acid + NAD+ + H 20 ->
• 0- and L- amino acid oxidases which oxidize a-keto acid + NADH + H+ + NH4+
amino acids to their corresponding keto • in the pentose phosphate pathway
acids;
• xanthine oxidase, involved in the conver- glucose-6-phosphate + NADP+ -> 6-
sion of purines to uric acid; phosphogluconolactone + NADPH + H+
• the flavoproteins of the mitochondrial elec- • in fatty acid synthesis
tron transport chain which links substrate
oxidation to the synthesis of ATP; RCOCH2COACP + NADPH + H+->
• succinate dehydrogenase, the enzyme RCHOHCH2COACP + NADP+
responsible for the conversion of succinate • in amino acid biosynthesis:
to fumarate in the TCA cycle;
• acyl-CoA dehydrogenases involved in the phenylalanine + NADPH + H+ ->
~-oxidation of fatty acids.
tyrosine + NADP+ + H 20
Because riboflavin is involved in many

Metabolism of nicotinic acid
important biochemical processes, it is not sur-
prising that its deficiency is reflected in a wide Nicotinic acid deficiency in humans results in
range of symptoms which vary from species to the development of the disease pellagra. This
species (see Table 8.2). disease is seen in populations that consume
o o
N~ NH H3C~VNH
H)~)CN ,.A"
HC
3 ..,. "
3 I
HC~N~~O
3 I
CH 2 CH 2
I I
HCOH HCOH
I I
HOOH HOOH
I I
HCOH HCOH 0
I I 11_
CHpH CHP-P-o
,-
o
Rlboftavln
Flavin mononucleotide
(FMN)
Flavin adenine dinucleotide

(FAD)
Figure 8.2 The structures of riboflavin and the coenzymes flavin mononucleotide (FMN) and flavin ade-
nine dinucleotide (FAD).
diets containing large amounts of maize. The mucosa. Both riboflavin and vitamin B6 are
deficiency can be overcome by dietary supple- required for this conversion, thus both affect
mentation with nicotinic acid, high-quality the efficiency of tryptophan use. Many ani-
protein or the amino acid tryptophan. The mals can synthesize sufficient nicotinic acid to
relationship between protein intake and nico- meet their requirements if the dietary protein
tinic acid results from the conversion of tryp- supply is sufficiently high in tryptophan.
tophan to nicotinic acid via kynurenine and Deficiency symptoms associated with nico-
quinolinic acid, probably in the intestinal tinic acid are summarized in Table 8.2.
Table 8.2 Summary of vitamin deficiencies in humans and animals
Vitamin Species Deficiency symptoms
Thiamin Humans 'Beri-beri': rapid weight loss, muscle weakness, loss of reflexes,
tachycardia, elevated blood pyruvate and lactate concentrations,
reduced erythrocyte transketolase activity
Poultry Polyneuritis: nerve degeneration, paralysis, head retraction
(opisthotonus), bradycardia
Pigs Loss of weight, weakness, enlargement of the heart, bradycardia
Ruminants Cerebrocortical necrosis (CCN), also called polioencephalomalacia
(PEM): muscle weakness, loss of reflexes, head retraction, bradycardia
Riboflavin Humans Seborrheic dermatitis around nose and mouth, soreness and burning
of lips and tongue, photophobia, corneal opacity
Poultry 'Curled-toe paralysis' associated with degenerative changes in the
myelin sheath, in severe cases causing pinching of sciatic nerve;
reduced egg production and poor hatchability
Pigs Thickened skin, scaly dermatitis, loss of hair, lens opacities and
cataracts
Ruminants In pre-ruminant animals: loss of appetite and hair, excessive salivation
and tear prodUction, lesions in the mouth and reddening of buccal
cavity
Nicotinic acid Humans 'Pellagra': dermal lesions and darkening of skin, nausea, vomiting and
diarrhoea
Cats and dogs Excessive production of thick saliva, oral lesions, ulceration and
bleeding of the digestive tract {'blacktongue' in dogs)
Pigs Loss of appetite and body weight, poor growth, rough or starring coat,
anaemia and degenerative changes of the digestive tract and nervous
system
Poultry Poor appetite and growth, red coloration of the crop, upper
oesophagus and mouth, dermatitis
Ruminants In pre-ruminant animals, deficiency leads to anorexia, diarrhoea and
dehydration
Pantothenic Humans Fatigue, headache, muscle weakness, 'burning feet syndrome'
acid Poultry Retardation of growth and feather production, dermatitis; eye lids
become granular and stick together; damage to the liver and spinal
cord
Pigs Scurvy skin and thin hair, a brownish secretion around the eyes,
gastrOintestinal disorders and slow growth; a characteristic symptom
is the locomotor disorder which leads to stiff, jerky, exaggerated
movement of the hind legs called 'goose-stepping'; post-mortem
examination often reveals signs of nerve degeneration, fat infiltration
of the liver and enlargement of the adrenals and heart
Ruminants In pre-ruminant animals, anorexia, reduced growth, rough coat,
dermatitis
Vitamin B6 Humans Seborrheic lesions around eyes, nose and mouth, anaemia, vomiting,
hyperirritability, convulsions
Poultry Poor growth and impaired plumage development, reduced egg
production and hatchability, unusual excitability, trembling and stiff
jerky movements eventually leading to convulsions
Pigs Anorexia, slow growth, anaemia and convulsions; degeneration of
peripheral nerves and deposition of a dark yellow pigment seen
post-mortem
Ruminants In pre-ruminant animals loss of appetite and, in severe cases, onset of
convulsions
Biotin Humans Dermatitis, somnolence, muscle pains, hyperaesthesia
Poultry Classical biotin deficiency causes irregular bone growth resulting in
leg abnormalities, particularly enlargement and deformation of the
hock joint (perosis); severe lesions of the underside of the feet can
occur; dry scaly skin is also found on the uppers ide of feet and legs,
beak growth is abnormal; fatty liver and kidney syndrome (FLKS) in
rapidly growing birds subjected to nutritional or environmental stress,
associated with reduced activity of hepatic pyruvate carboxylase
which limits the ability to synthesize glucose when the dietary supply
of glucose is disrupted; increase in tissue palmitoleic acid (C16:1)
content and a decrease in a number of the PUFAs probably caused by
a reduced availability of malonyl-CoA for fatty acid elongation - birds
affected by FLKS rarely show classical signs of biotin deficiency
Pigs Dermatitis characterized by shedding of large pieces of skin ('greasy
maize flakes'); a characteristic soft hoof condition seen in pigs is
'concrete disease' so-called because the feet of animals kept on
abrasive surfaces such as concrete develop severe cracking, often with
secondary infections
Ruminants No clear link between biotin status and lameness
Horses Cracked and weak hooves
Rats and dogs Ascending paralysis, cessation of growth and a spectacled eye
condition
Folic acid All species Anaemias, typically megaloblastic anaemia in which the red blood
cells are large and immature; leucopenia, a condition in which the
number of white blood cell declines; increased urinary excretion of
formiminoglutamic acid (FIGLU)
Vitamin B12 Humans Pernicious anaemia, weakness, lethargy and progressive paralysis;
urinary excretion of FIGLU is elevated following a histidine loading
test; increased excretion of methylmalonic acid
Ruminants Poor growth, anaemia characterized by the presence of megaloblasts
and pigs and, in severe cases, paleness of the mucus membranes; lack of
posterior coordination and unsteadiness of gait; urinary excretion
of FIGLU is elevated following a histidine loading test and is often
accompanied by urinary excretion of methylmalonic acid -
marginal vitamin B12 deficiency is difficult to detect and is typified by
animals that are described as 'unthrifty'; in ruminants, similar
symptoms can arise due to cobalt deficiency
Vitamin C Humans, other 'Scurvy': swollen, bleeding and ulcerated gums, loose teeth, weak
primates and bones, fragile capillaries leading to widespread haemorrhaging,
guinea pigs increased susceptibility to infection
Pigs and Neonates unable to synthesize this vitamin in the first few weeks of
ruminants life, and may show increased susceptibility to infection if milk supply
is inadequate
Poultry Deficiency not normally seen; in heat-stressed birds supplementation
can increase growth, egg quality and hatchability
Choline Humans Possible liver damage
Other species Fatty infiltration of the liver, necrosis or haemorrhagic lesions of the
liver, kidney and joints
Carnitine All species Elevated levels of plasma non-esterified fatty acids and triacylglycerols
suggesting impaired fatty acid oxidation and redirection of fatty acids
to alternative pathways of metabolism; in severe cases the increase in
intracellular concentrations of acyl-CoAs may impair the function of
other metabolic pathways leading to liver dysfunction
Vitamin A Humans Night blindness, xerophthalmia, follicular hyperkeratosis
Pigs Uncoordination, spasm and paralysis; failure of oestrus, foetal and
neonatal deformities, foetal resorption
Poultry Staggering gait, ruffled plummage, reduced egg production and
hatchability, keratinization of mucus epithelium
Ruminants Nightblindness, xerophthalmia; abnormal bone remodelling leading to
blindness and deafness; low conception rates, foetal resorption and
retained placenta; keratinization of digestive, respiratory and
reproductive tract epithelium; reduced sperm viability
Vitamin D Humans Rickets in growing children, osteomalacia in adults
Pigs Rickets in growing animals, osteomalacia in mature animals
Poultry Rickets, soft beak and claws, reduced egg production and hatchability,
thin egg shells
Ruminants Rickets in growing animals, osteomalacia in mature animals; reduced
milk production and inhibition of oestrus
Vitamin E Humans Haemolytic anaemia in infants, increased erythrocyte haemolysis in
adults; few other symptoms
Pigs Nutritional microangiopathy characterized by haemorrhagic lesions of
the heart, also known as 'mulberry heart disease'; hepatic necrosis
Poultry Exudative diathesis (subcutaneous oedema resulting from increased
capillary permability); encephalomalacia ('crazy chick disease');
muscular dystrophy
Ruminants Muscular dystrophy affecting both skeletal and cardiac muscle (,white
muscle disease' in cattle, 'stiff lamb disease' in sheep
Vitamin K All species Reduced blood prothrombin, increased blood clotting time; in farm
animals, extensive subcutaenous and internal haemorrhaging and
anaemia; in ruminants' sweet clover disease' due to presence of
metabolic antagonist, dicumarol, in mouldy sweet clover
8.1.5 PANTOTHENIC ACID gastrointestinal disorders and lesions of the

nervous system (see Table 8.2).
Pantothenic acid (Figure 8.3) is a constituent of
8.1.6 PYRIDOXINE, PYRIDOXAL AND
coenzyme A and of acyl carrier protein (ACP),
PYRIDOXAMINE (VITAMIN B6)
both of which contain the same functional
group, 4' -phosphopantotheine, formed from This vitamin exists in three forms which are
pantothenic acid and l3-mercaptoethylamine. interconvertible in body tissues. The parent
The terminal SH group of 4' -phosphopan- compound is pyridoxine which is converted to
totheine forms a thioester bond with car- pyridoxal and pyridoxamine (Figure 8.3).
boxylic acids to produce coenzyme A or ACP
derivatives. One coenzyme A derivative,
acetyl-CoA, occupies a particularly important
role in intermediary metabolism (see Chapters Pyridoxal phosphate and pyridoxamine phos-
11 and 19). phate act as coenzymes in many enzymes of
Pantothenic acid has a variety of metabolic amino acid metabolism, e.g. transaminases,
functions, and deficiency results in a wide amino acid deaminases and amino acid decar-
range of symptoms including growth and boxylases. In transaminase reactions, pyridox-
reproductive failures, skin and hair lesions, al phosphate acts as the carrier of the amino
~COOH
~..N~
Nicotinic acid Nicotinamide
CH 0
I 3 II
CH--C--CH-C--NH-CH-CH-COOH
3 I I 2 2
CH 3 0H
Pantothenic acid
CH 20H CHO
HOCH~OH HOCH~OH
~.~
N CH 3
~.~
N CH 3
Pyridoxine Pyridoxal Pyridoxamine
o
HNJlNH
ttCH-CH-CH-CH'COOH
2 2 2 2
Biotin
Figure 8.3 The structures of nicotinic acid, nicotinamide, pantothenic acid, pyridoxine, pyridixal, pyri-
doxamine and biotin.
group as it is transferred from an amino acid to • non-oxidative decarboxylation reactions:

an a-ketoacid. In so doing it is transformed to
pyridoxamine phosphate. histidine -+ histamine + CO 2
• transaminase (aminotransferase) reactions: Vitamin B6 has a number of other important

biochemical functions. For example:
L-alanine + a-ketoglutarate -+
pyruvate + L-glutamate • in the synthesis of neurotransmitters adren-
aline, noradrenaline, dopamine, histamine
• non-oxidative deamination reactions:
and l'-aminobutyric acid;
serine -+ pyruvate + NH3 • in the synthesis of protoporphyrin IX found
threonine -+ a-ketobutyrate + NH3 in haemoglobin and myoglobin;
• in glycogenolysis where it is a coenzyme for carbon number 9 to a p-aminobenzoyl residue
glycogen phosphorylase; which is, in turn, linked to poly-')'-glutamate,
• in the formation of nicotinic acid from tryp- where the number of glutamate residues
tophan. varies up to a maximum of eight. The term
folic acid or folate is commonly used to mean
Because many enzymatic reactions are anyone of the various conjugated (glutamy-
dependent on vitamin B6 a wide variety of lated) forms, although by strict definition folic
abnormalities are observed in the deficient acid contains only one glutamate residue. The
animal (see Table 8.2). pteridine ring is usually in a reduced form,
with hydrogens on carbons 6 and 7 and nitro-
8.1.7 BIOTIN gens 5 and 8 giving tetrahydrofolic acid.
Biotin (Figure 8.3) functions as a coenzyme in
carboxyl group transfer reactions. In addition, Folic acid has a number of coenzyme forms
biotin may have functions in the synthesis of which occur mainly as polyglutamates in ani-
proteins, particularly keratins, which cannot mal tissues. The main functions of tetrahydro-
be explained entirely on the basis of its role in folic acid are in the transfer of one-carbon
carboxylation reactions. units in degradative and biosynthetic reac-
tions. The coenzyme forms found in tissues
are 5-methyl-tetrahydrofolate; 5,1O-methyl-
Metabolism of biotin ene-tetrahydrofolate; 5,10-methenyl-tetrahy-
Biotin is found in nature both as the free form drofolate; 5-formyl-tetrahydrofolate; 10-
and in a form bound to an amino acid, usually formyl-tetrahydrofolate and 5-formimino-
lysine (biocytin). In animals, it is readily tetrah ydrofola te.
absorbed from the small intestine in either the Some of the processes which require the
free or bound form. Little is known about the transfer of one-carbon groups mediated by
metabolism of biotin in humans or animals. tetrahydrofolate are:
Several biotin-binding proteins are produced • glycine, histidine and tryptophan degrada-
by microorganisms, e.g. streptavidin and stra- tion;
vidin from Saccharomyces avidinii. Egg white • conversion of serine to methionine;
contains a similar biotin-binding protein, • synthesis of inosine, a precursor of purines,
avidin. Administration of these binding pro- adenine and guanine;
teins by mouth greatly reduces the availability • synthesis of the pyrimidine thymidine
of dietary biotin, apparently by preventing (Figure 8.4).
absorption across the intestinal mucosa.
Deficiency can easily be -induced in most
Metabolism of folic acid
animals by feeding raw egg white. Symptoms
of biotin deficiency common to all species are Dietary folic acid occurs mainly as polygluta-
retarded growth, dermatitis, loss of hair and mates. The vitamin is absorbed in the duode-
disturbances of the nervous system (see Table num and the jejunum, apparently by an active
8.2). process which is stimulated by glucose. During
passage through the intestinal mucosa, the
polyglutamate derivatives are hydrolysed to
8.1.8 FOLIC ACID
the monoglutamate and may undergo reduc-
The structure of folic acid is shown in Figure tion and methylation to produce 5-methyl-
8.4. It consists of a pteridine ring linked via tetrahydrofolate. These absorbed forms enter
(a)
Folic acid
5,6,7,8-tetrahydrofolic acid
(THF)
(b)
. ~ J-':~
N5 10-methylene-THF
Thymidylate synthetase
o
N~CH3
O~Nj
I I
deoxyribose deoxyribose
deoxyuridine deoxythymidine
diphosphate diphosphate
Figure 8.4 The structures of folic acid and tetrahydrofolic acid (THF). Folic acid contains a single gluta-
mate residue. Tetrahydrofolate and its derivatives can occur in the polyglutamate form in which n may
= 1-8. The conversion of deoxyuridine diphosphate (dUDP) to deoxythymidine diphosphate (dTDP)
showing the transfer of a one-carbon unit from Ns,lO- methylene-THF.
the blood stream and are transported to the 8.1.9 VITAMIN B12
liver and peripheral tissues, Mammalian tis-
sues can synthesize the pteridine ring and Vitamin B12 has the most complex structure of
polyglutamate but are unable to link these all vitamins (Figure 8.5) and was the last to be
together through p-aminobenzoic acid. isolated and identified. The term vitamin B12 is
Deficiency symptoms are mainly associated a generic term used to refer to a group of com-
with abnormalities in the formation of red and pounds characterized by the presence of a
white cells and disturbances of nucleic acid tetrapyrrole ring, in which the inner nitrogen
synthesis (see Table 8.2). atom of each pyrrole ring is coordinated to a
single atom of cobalt. The commercially avail- tions is known to occur. This is the conversion
able form of vitamin B12 is cyanocobalamin, in of methylmalonyl-CoA to succinyl-CoA
which a cyanide group is attached to the (Figure 8.6). This reaction occurs in all animals
cobalt atom. Cyanocobalamin, although natu- and is a useful route by which propionate
rally occurring, is not an important form of the derived from odd-numbered fatty acid oxida-
vitamin. In the three most common forms tion and amino acid catabolism can be direct-
found in tissues and feedstuffs, the cyanide ed to glucose synthesis. It is of particular
group is replaced by a 5' -deoxyadenosyl importance in ruminants in which little or no
group to produce 5' -deoxyadenosyl cobalamin dietary glucose is absorbed from the digestive
or coenzyme B12; by a methyl group to pro- tract, and glucose supply is largely dependent
duce methylcobalamin; or by a hydroxyl on gluconeogenesis from compounds such as
group to produce hydroxocobalamin. propionate and amino acids.
Only one other vitamin Bl2-dependent
reaction occurs in mammalian tissues. In this
reaction, catalysed by 5-methyl-tetrahydrofo-
In microorganisms, 5' -deoxyadenosylcobal- late homocysteine methyl transferase, methyl-
amin is involved in a number of rearrange- cobalamin acts as a methyl group carrier in
ment reactions. However, in animals only one the conversion of homocysteine to methion-
of this type of vitamin B12-dependent reac- ine (Figure 8.6).
CH 2
"0 - «
I
CHCH 3 N : ( XCH 3
I"
I
, ,..0 N CH
o..",P"0 HO
~f--{~
~,,~
HOCH 0 H
2
R=CN Cyanocobalamin (Vitamin 812)

R=OH Hydroxocobalamin (Vitamin 812.)
R=CH3 Methylcobalamin (methyl 812)
R = 5'-deoxyadenosine 5' -deoxyadenosylcobalamin (coenzyme 812)
Figure 8.5 Vitamin BI2 and its derivatives.

COSCoA
CH3
I
CHCOSCoA
Coenzyme 8'2 ~
I
COOH
MethylmalonyI-CoA mutase
SH
I
CH 3 N5-MethyiTHF THF
I
CHa
I
CH-NH 2
I
COOH
5'-methyltetrahydrofolate
Homocysteine homocysteine methyltransferase Methionine
Figure 8.6 The conversion of methylmalonyl-CoA to succinyl-CoA and homocysteine to methionine.
Metabolism of vitamin BIZ Biochemical functions

The vitamin is relatively slowly absorbed In humans, a deficiency of vitamin C is classical-
from the small intestine and its uptake is an ly linked with the development of the disease
active process dependent on the presence of scurvy, in which connective tissue synthesis is
a glycoprotein called intrinsic factor which is impaired. Amino acid analysis of collagen, the
produced by the parietal cells of the stomach principal protein in connective tissue, shows
(abomasum). Once absorbed into the blood that it contains a significant proportion of the
stream it is attached to specific transport pro- amino acid hydroxyproline. This amino acid is
teins called transcobalamins. Transfer of the synthesized from proline by an ascorbate-
vitamin from transcobalamins to the intracel- dependent hydroxylation reaction. Ascorbic
lular location is receptor-mediated. Within acid is also involved in the hydroxylation of
the cell most vitamin B12 is found in associa- lysine. The resulting hydroxylysine is further
tion with the two vitamin B l2-dependent glycosylated and forms cross-links between col-
enzymes, methylmalonyl-CoA mutase and 5- lagen fibres.
methyl-tetra hydro folate methyltransferase. A number of other hydroxylation reactions
Deficiency symptoms associated with vita- require ascorbic acid, for example:
min B12 are listed in Table 8.2.
• the ascorbate-dependent copper-contain-
ing enzyme dopamine J3-hydroxylase catal-
8.1.10 VITAMIN C
yses the hydroxylation of dopamine in the
The active forms of vitamin C are the L iso- synthesis of noradrenaline;
mers of ascorbic and dehydroascorbic acid • two ascorbate-dependent enzymes, tri-
shown in Figure 8.7. methyllysine hydroxylase and ),-butyro-
Ascorbic acid Dehydroascorbic acid
(CH 3hN+ ~H-CH-CH-COOH

2 I 2
OH
Choline
Carnitlne
Figure 8.7 The structures of ascorbic acid, dehydroascorbic acid, choline and carnitine.
betaine hydroxylase are required for the Metabolism of vitamin C

synthesis of carnitine;
Vitamin C can be synthesized by all microor-
• ascorbic acid is a coenzyme for peptidyl
ganisms and plants, and most animals. The
glycine hydroxylase required for the amida-
process involves the conversion of hexose sug-
tion of a number of neuropeptides.
ars (mainly glucose and galactose) to ascorbic
Other roles of ascorbic acid relate to its abil- acid. Certain insects, fish, birds, flying mam-
ity to act as a reducing agent and antioxidant. mals, guinea pigs and primates lack L-gulono-
Dietary ascorbate has beneficial effects on iron lactone oxidase, the penultimate enzyme in
absorption from the small intestine. Dietary this pathway, and are dependent on an
iron is absorbed in the Fe (II) state and not as endogenous supply.
Fe(III). The presence of ascorbate ensures that The requirement for vitamin C changes
iron is maintained in its reduced state, and with physiological and health status due to
has the added effect that it acts as a chelating increased turnover of the vitamin. For exam-
agent thereby increasing the efficiency of iron ple, increased metabolism occurs during bac-
absorption. As an antioxidant ascorbate has terial infection when vitamin C is required to
two roles. Firstly, it protects against losses of neutralize the free radicals produced by
the lipid-phase antioxidant, vitamin E (a-toco- phagocytes to kill microorganisms.
pherol), by promoting the regeneration of a- Typical deficiency symptoms associated
tocopherol from the a-tocopheryl radical pro- with vitamin C are listed in Table 8.2
duced during peroxidation of unsaturated
fatty acids. Secondly, ascorbate is an impor-
8.1.11 CHOLINE
tant aqueous-phase antioxidant, providing
protection against free radical species in the Choline (Figure 8.7) is commonly classified as
cytoplasm. a vitamin, although it does not entirely con-
form to the classical definition of a vitamin as 8.1.12 CARNITINE
it can be synthesized by most animal species, is
The structure of carnitine (3-hydroxy-4-N-
required in larger quantities than most vita-
trimethylaminobutyric acid) is shown in
mins, and has a structural rather than a coen-
Figure 8.7. Like choline, carnitine does not fit
zyme function.
the classical definition of a vitamin.
Biochemical functions Biochemical functions

Choline has three main biochemical functions. Carnitine is required for the oxidation of fatty
• It is a part of the phospholipids phos- acids described in detail in Chapter 13. Fatty
phatidylcholine and sphingomyelin, both acids destined for l3-oxidation are activated in
of which are structural components of bio- the cytoplasm to yield fatty acyl-CoAs. As
logical membranes. The details of the syn- fatty acyl-CoAs cannot cross the inner mito-
thesis of these lipids are described in chondrial membrane, they are first converted
Chapter 19. to fatty acyl-carnitines which are transported
• It is required for the synthesis of the neuro- into the mitochondrial matrix via a specific
transmitter acetylcholine. membrane translocase, prior to a second
• It is a source of methyl groups for trans- exchange reaction which regenerates fatty
methylation reactions, and serves as a acyl-CoAs. This is the only known function of
source of betaine which performs a similar carnitine in animal tissues.
function.
Metabolism of carnitine
Metabolism of choline Carnitine is present in food of animal origin
but little or no carnitine is found in plant
Choline may be present in the diet both in the
foods. Little is known about its digestion or
free form and as phosphatidylcholine. In the
bioavailability and it is assumed that it is read-
free form about two-thirds is converted to
ily absorbed across the small intestine. The
trimethylamine by the intestinal microflora
major sites of synthesis in animal tissues are
and excreted via the urine. The remaining
the liver and kidney. The main precursor for
one-third is absorbed directly into the blood
synthesis is the amino acid lysine. This path-
s~ream via the jejunum and ileum. The diges-
way is interesting in that it starts from protein-
tion of phosphatidylcholine is described in
incorporated lysine and not free lysine. The
Chapter 28.
lysine residue undergoes transmethylation to
Synthesis of phosphatidylcholine and
form trimethyllysine which is then released by
sphingomyelin occurs in most tissues, as
proteolysis. The subsequent conversion of
described in Chapter 19. Conversion to acetyl-
trimethyllysine to carnitine involves two
choline is a highly active process in nervous
hydroxylation steps, both of which are ascor-
tissue and is catalysed by the enzyme choline
bic acid-dependent. Dietary deficiency of
acetyltransferase (CAT).
lysine, methionine or ascorbic acid may there-
choline + acetyl-CoA --> acetylcholine + CoA fore increase the requirement for carnitine
supplementation.
Betaine synthesis is active in many tissues,
particularly in the liver and kidney, and is
8.1.13 VITAMIN A
used in the transmethylation of homocysteine
to methionine by a reaction which does not Vitamin A exists in three forms: retinol, retinal
require vitamin B12 or folic acid. (retinaldehyde), and retinoic acid. The struc-
tures shown in Figure B.B are the all-trans iso- the most important source of vitamin A is 13-
mers which are quantitatively the most impor- carotene (see Figure B.B).
tant forms. Vitamin A does not occur in plant
materials but is present in its precursor forms,
the carotenes. There are a number of different
type of carotene and carotene derivatives, Vitamin A has an important role in vision. It
some of which have considerable potency as also appears to function as a regulator of the
vitamin A precursors; however, quantitatively differentiation and metabolism of cells. Its role
Retinol
CHO
Retinal (Retinaldehyde)
COOH
Retlnolc acid
p-Carotene
Figure 8.8 The structures of retinol, retinal (retinaldehyde), retinoic acid and j3-carotene.
in visual processes is now well characterized. reductase. Most retinol is then esterified to
Its other functions are less well understood. palmitic acid and incorporated into chylomi-
crons which are released into the blood stream
via the lymphatic system. The ability to trans-
Vitamin A and vision
fer absorbed dietary carotenoids from the
The retina contains cells called rods and cones. intestinal mucosa to blood and the tissues
The former are concerned with vision in dim varies from species to species. For example,
light, while the latter are involved in vision in humans, birds and ruminants can absorb both
bright light and are responsible for colour vitamin A and the carotenoids whereas pigs
vision. The characteristic pigments of rods and and rats can absorb only vitamin A.
cones are conjugated carotenoid proteins Vitamin A is usually present in foods in an
known, respectively, as rhodopsin and esterified form as retinyl palmitate. These
iodopsin. They differ only in the opsin or pro- esters are hydrolysed in the lumen of the small
tein moieties. In rods, opsin (an intrinsic trans- intestine. Free retinol is absorbed by the intesti-
membrane protein) makes up about 90% of nal mucosa where it is usually re-esterified to
the proteins in the specialized disk mem- palmitic acid and incorporated into lymph
branes. The carotenoid common to both lipoproteins for transport to the blood stream.
rhodopsin and iodopsin is ll-cis-retinal. Retinyl esters are stored mainly in the liver.
Rhodopsin, which is essential to night Absorbed carotenoids are stored in liver and
vision, is a bright red pigment. On exposure to adipose tissue. The presence of carotenoids
light it bleaches, resulting in the conversion of gives rise to a yellow coloration of the fat
ll-cis-retinal to all-trans-retinal (a yellow com- depots. During mobilization of stored vitamin
pound) and its release from the membrane- A from the liver, retinyl esters are hydrolysed
bound protein opsin. All-trans-retinal is enzymatically. The retinol is transferred to a
reduced by alcohol dehydrogenase to the retinol-binding protein (RBP) prior to release
colourless all-trans-retinol. The latter is isomer- into the blood stream for distribution to other
ized to ll-cis-retinol, reduced by alcohol dehy- tissues. Uptake of RBP-bound retinol is a
drogenase to ll-cis-retinal and, in the dark, membrane receptor-mediated process. Once
re-incorporated into rhodopsin (Figure 8.9). absorbed into the cell the retinol is rapidly
During these transformations some vitamin A transferred to cellular retinol-binding proteins
is lost. Thus a deficiency of the vitamin even- (CRBPs) which may serve to protect the
tually results in the inability to see in dim light retinol from oxidation and direct its further
and the development of the condition com- metabolism.
monly known as night blindness, the earliest
symptom of vitamin A deficiency in humans.
Deficiency symptoms
Numerous deficiency symptoms have been
Metabolism of vitamin A
described in humans and farm animals. In
Both vitamin A and ~-carotene are absorbed general these fall into three main areas: visual
from the small intestine with the fat fraction of disorders; abnormalities in cell growth and
the diet. Conditions that impair fat absorption differentiation; and keratinization of epithelial
decrease the absorption of vitamin A and ~ tissues (see Table 8.2).
carotene. The majority of the ~-carotene
absorbed by the intestinal mucosa is cleaved
8.1.14 VITAMIN D
by the enzyme 15,15' -~-carotene dioxygenase
to yield two molecules of retinal which are Vitamin 0, sometimes called the sunshine vit-
then reduced to retinol by retinaldehyde amin, is primarily known as the vitamin which
>-Rh~~
Retinal (11-c;s) Retinal (all-trans)
Opsin
oxidation by reduction by
alcohol dehydrogenase alcohol dehydrogenase
Retinol (11-c;s) Retinol (all-trans)
Figure 8.9 The visual cycle.
prevents the development of rickets. There are from bone. It is essential for the normal calcifi-
two forms of vitamin D, ergocalciferol (vita- cation of bone. An adequate supply of vitamin
min D2) and cholecalciferol (vitamin D3) and, D is particularly important in young animals
although it has been shown that about 10 where bone growth is rapid. It is less impor-
sterol derivatives have some vitamin D activi- tant in adult animals where bone mass is rela-
ty, these are the two important dietary sources tively stable, but females are particularly sus-
of the vitamin (Figure 8.10). ceptible to changes in vitamin D status during
pregnancy and lactation.
The mode of action of 1,25-dihydroxy vita-
min D is not yet fully understood. It is thought
The most important functions of the active that in the small intestine the vitamin binds to
metabolite of vitamin D, 1,25-dihydroxychole- receptor sites in the plasma membrane, and
calciferol, are in the regulation of calcium and that the vitamin-receptor complex is trans-
phosphorus metabolism. It stimulates the syn- ported to the nucleus, where it initiates the
thesis of a calcium-binding protein in the production of mRNA which codes for the syn-
intestinal epithelium which promotes the thesis of the calcium-binding protein cal-
absorption of dietary calcium, acts on the dis- bindin-D. In many respects the action of the
tal renal tubule to increase resorption of calci- vitamin is very similar to that of the steroid
um, and stimulates the mobilization of calcium hormones, and it has been suggested that 1,25-
HO
7 -Dehydrocholesterol Ergosterol
UV light UV light
HO
Vitamin 03 (cholecalciferol) Vitamin 02 (ergocalciferol)
Figure 8.10 The conversion of 7-dehydrocholesterol and ergosterol to vitamin D3 (cholecalciferol) and D2
(ergocalciferol), respectively, under the influence of ultraviolet light.
dihydroxy vitamin 0 should be reclassified as enhances the absorption of fat influences the
a hormone and not a vitamin. ability of animals to absorb the sterol precur-
sors or the vitamin. The body has some ability
to store the vitamin in liver and adipose tissue
Metabolism of vitamin 0
but to a much lesser extent than vitamin A.
The sterols 7-dehydrocholesterol and ergos- The effect of sunlight on the body is an
terol are important precursors of vitamin D. important factor in the production of vitamin
Both the precursors and the vitamin itself O. Skin and sebaceous secretions contain 7-
(cholecalciferol and ergocalciferol) are dehydrocholesterol and some ergosterol of
absorbed in the small intestine and, like vita- dietary origin. These sterols are converted to
min A, any factor which interferes with or vitamin 0 3 and O2, respectively, by exposure
to ultraviolet light. The vitamins produced plete calcification of the bones. Bone forma-
may be absorbed directly through the skin or, tion proceeds via mineralization of the organ-
in the case of many animals, licked from the ic cartilage matrix. Analysis of rachitic bones
skin and absorbed through the gastrointesti- reveals a decreased concentration of calcium
nal tract. Irradiation is less effective in dark- and phosphorus leading to a reduction in
pigmented skin or through a heavy coat of physical strength. In young, rapidly growing
hair. The effectiveness of irradiation depends animals the bony framework of the body is
on the wavelength and intensity of the ultra- unable to support the growing muscle mass.
violet light incident on the body. Thus, it is This is particularly pronounced in the long
most effective in the tropics, in the summer, at bones which tend to remain soft and bend
noon and at high altitudes. These variations under the weight of the body. The principal
are of great importance in vitamin D nutrition. cause of rickets is not a defect in the process of
Animals which are at pasture during the sum- calcification but rather a lack of calcium, the
mer never suffer from deficiency even though major substrate for the mineralization of the
their diet is practically devoid of the vitamin. bone matrix. Bone is a dynamic tissue and
This is not the case in winter in temperate cli- although bone growth is greatest in the early
mates, when animals may be outside only for stages of life, even in adult animals bone is in
short periods, there are generally fewer sunny a constant state of turnover. Lack of vitamin D
days and the sunlight is less intense. Thus dur- in adult life leads to a loss of calcium from the
ing winter months in the temperate regions of bone matrix resulting in the condition known
the world it is necessary to supplement live- as osteomalacia.
stock feeds.
Only recently has it become clear that vita-
8.1.15 VITAMIN E
min D is further metabolized. The first step
occurs in the liver where the vitamin is hydrox- Vitamin E is the generic term used to describe
ylated by a-25-hydroxylase to produce 25-0H- a group of related compounds, the toco-
vitamin D, which is more active than the unhy- pherols and tocotrienols. Each exists in a num-
droxylated form. The second step occurs in the ber of isomeric forms designated a, 13, 'Y and 0
kidney where a second hydroxylation pro- isomers. The a isomers are shown in Figure
duces 1,25-dihydroxy-vitamin D. This is the 8.11. The biopotency of the isomers is
most active form of the vitamin. The kidney expressed in relation to a-tocopherol which is
also contains a 24-hydroxylase which catalyses the most abundant and active form.
the formation of 24,25-dihydroxy-vitamin D.
The functions of this metabolite are still unclear.
It was initially thought to be a mechanism of
excretion of excess vitamin D; however, recent The vitamin was first known as the antisterility
research suggests that it has a role in bone min- vitamin following studies carried out in rats;
eralization and may suppress the secretion of however, the relationship between vitamin E
parathyroid hormone. Elimination of vitamin D and fertility does not hold for all species.
from the body requires conversion of 1,25-dihy- Vitamin E deficiency has never been linked
droxy vitamin D to 1,24,25-trihydroxy vitamin with fertility problems in humans or ruminants.
D, followed by formation of more polar deriva- Perhaps the most important function of a-
tives in the liver, transfer to bile, and excretion tocopherol in plant and animal tissues is as an
via the faeces. antioxidant. It is found in membranes in close
The primary symptom of vitamin D defi- association with PUFAs, which are particularly
ciency is rickets, which is caused by incom- susceptible to oxidation. Peroxides arise due to
a-Tocopherol
a-TocoIrIenoI
o
o
VItamin K1 (Phylloquinone)
«r o
o
eH
,
VItamin K3 (Menadione) Warfarin
OH H~OH
~ :-...
I
o
2 I
....
Dicoumarol
Figure 8.11 The structure of a-tocopherol; a-tocotrienol; the various forms of vitamin K: vitamin KI (phyl-
loquinone), vitamin Kz (menaquinone), vitamin ~ (menadione); warfarin; and dicoumarol.
the reaction of PUFAs with reactive oxygen such as a-tocopherol acetate, are hydrolysed
species such as superoxide (° 2 and hydroxyl
0
-) prior to absorption, probably by mucosal
(OHO) radicals. They also occur as intermediates esterase. Unlike retinol, there does not appear
in the conversion of PUFAs to eicosanoids. to be a specific transport protein for a-toco-
Lipid peroxides are unstable, and spontaneous- pherol which is found mainly in the lipopro-
ly decompose to form free radicals which initi- tein fraction. a-Tocopherol is the major form
ate an autocatalytic cycle of membrane-lipid of vitamin E deposited in tissues: major
oxidation. If uncontrolled, this process can have reserves are found in liver, adipose tissue and
damaging effects on membrane structure and muscle. There is very little further metabolism
function in vivo and can lead to development of of the vitamin.
rancidity in lipid-rich foods such as meat and Vitamin E deficiency results in a wide range
oils. a-Tocopherol prevents uncontrolled lipid of symptoms in farm animals but is poorly
peroxidation by acting as a chain-breaking characterized in humans except in the
antioxidant which reacts with peroxide-propa- neonate, particularly the pre-term infant. The
gating radicals and is converted to the relative- main effects of deficiency are listed in Table
ly stable a-tocopheroxyl radical. 8.2. Because its antioxidant function is part of
The antioxidant properties of a-tocopherol an integrated antioxidant defence mechanism
are important in agricultural food products. including glutathione peroxidase (a selenium-
The shelf life of meat can be extended by containing enzyme), many of the symptoms of
increasing its a-tocopherol content, which vitamin E deficiency can be induced by seleni-
slows the process of tissue fatty acid oxida- um deficiency and alleviated by selenium sup-
tion and delays the development of 'off plementation.
flavours' and taints. In addition, dietary sup-
plementation with supranutritional levels of
8.1.16 VITAMIN K
a-tocopherol inhibits the oxidation of meat
pigments, principally myoglobin, thereby Vitamin K occurs naturally in two main forms,
maintaining the fresh meat colour without vitamin KJ (phylloquinone), found in plants,
the need to add artificial colorants. and vitamin Kz (menaquinone) present in bac-
There is increasing interest in the role of a- teria and animals. A third compound, mena-
tocopherol in the immune response. dione, has some vitamin K activity and is
Lymphocytes and mononuclear cells have sometimes referred to as vitamin~. Vitamins
been shown to contain the highest concentra- KJ and Kz contain phytyl and polyisoprenoid
tion of a-tocopherol of all the cells in the body. side chains, respectively (Figure 8.11).
Experiments have shown that supplementa-
tion of animal diets with a-tocopherol results
in an increase in lymphocyte transformation
in response to mitogen stimulation, increased Vitamin K is important in regulating the syn-
antibody production and greater resistance to thesis of calcium-binding proteins containing
pathogenic organisms. Some of these effects 'Y-carboxyglutamic acid. Some of these are
may be mediated by the influence of a-toco- important in blood clotting and have been
pherol on eicosanoid metabolism and by identified as factors II (prothrombin), VII, IX
changes in membrane fluidity. and X. The synthesis of prothrombin involves
the post-translational modification of a pre-
cursor protein. In vitamin K deficiency this
Metabolism of vitamin E
precursor undergoes incomplete glutamate
Tocopherols are absorbed via the small intes- carboxylation, resulting in the production of
tine and transported to the blood stream via preprothrombin. However, in animals with
the lymph system. Any esters of vitamin E, adequate vitamin K, the precursor is converted
Minerals in biochemistry 127
to prothrombin which contains ten "{-carboxy- functions in animals are described; their role
glutamic acid residues in the 40 amino acids at in plants is considered in Chapter 26. Animals
the amino-terminal end. The precise role of vit- require some elements in quite large quantities
amin K in the enzyme mechanism is still not where they may have a structural role as well
clear; however, in all species a deficiency of this as important biochemical functions. These are
vitamin leads to a fall in prothrombin concen- referred to as the major mineral elements. It is
tration in blood and the incidence of wide- the convention to consider calcium, chlorine,
spread haemorrhaging (Table 8.2). Vitamin K magnesium, phosphorus, potassium, sodium
may have a similar role in skeletal tissue where and sulphur as the major mineral elements.
the protein osteocalcin also contains ,,{-car- These are usually present in tissues in quanti-
boxyglutamic acid. This protein appears to ties varying from 15 g kgl tissue for calcium to
have a role in the mineralization of bone and is 0.4 g kgl for magnesium.
present in higher concentrations in rapidly The remaining mineral elements are usual-
growing animals. ly referred to as trace elements. As analytical
A number of analogues and antagonists to techniques become more sensitive the list of
vitamin K have been developed for medical trace elements present in living tissues is
and pest-control purposes. The best known of growing. Some of these trace elements have
these are a family of coumarin derivatives clearly identified biochemical functions: thus
which have been used successfully in antico- it is clear that cobalt, copper, iodine, iron, man-
agulation therapy. One of the most widely ganese, molybdenum, selenium and zinc are
used vitamin K antagonists is the rodenticide involved in specific biochemical processes.
warfarin. There is concern that rat popula- Other trace elements such as nickel, chromi-
tions are now developing resistance to the um and vanadium have metabolic roles that
effects of warfarin, which has led to the devel- are only now being discovered. The concen-
opment of a number of other potent tration of trace elements varies considerably,
coumarin-based rodenticides. from iron, which may be present in tissues at
between 20 and 80 mg kgl tissue, to cobalt
present at concentrations as low as 0.02-0.1
Metabolism of vitamin K mg kg l. Some elements may be toxic at high
The main site of absorption is the small intes- concentrations. In some cases, such as copper,
tine, and the vitamin is transported to the molybdenum and selenium, there is a fine line
blood stream via the lymph. There is little evi- between normal and toxic concentrations, and
dence of any specific carrier protein for trans- the toxic concentration may show consider-
port in the blood, and most vitamin K in blood able animal species variation. For example,
is associated with the lipoprotein fraction. levels of copper normally included in pig diets
Bacteria can synthesize menaquinone so that would be toxic if fed to sheep.
ruminants, and those non-ruminants with
well developed caeca, can synthesize much of 8.2.2 CALCIUM
their requirements. There is little storage of the
vitamin in body tissues, but it is found in high- Calcium is the most abundant mineral found
est concentrations in the liver. in vertebrates. Bone constitutes the major
body reserve which can be mobilized when
calcium intake is low.
8.2 MINERALS IN BIOCHEMISTRY In addition to its structural role, calcium has
numerous metabolic functions. It acts as an
8.2.1 INTRODUCTION
important intracellular messenger involved in
Many mineral elements have important func- the regulation of a number of enzymes. For
tions in living organisms. In this chapter their example, in contracting muscle the calcium
activation of glycogen phosphorylase, pyru- phosphorylated nucleotides, ATP and GTP. It
vate dehydrogenase, isocitrate dehydrogenase is a component of nucleic acids, phosphopro-
and a-ketoglutarate dehydrogenase serves to teins and phospholipids. The phosphorus in
increase glycogen breakdown and glucose oxi- vertebrate bone constitutes more than 80% of
dation to provide the ATP required for muscle the total body phosphorus, and acts as a useful
contraction. Many of the functions of calcium store which can be used to buffer phosphorus
are mediated via the multipurpose calcium- requirements during periods of dietary insuf-
binding protein calmodulin, and may be initi- ficiency.
ated hormonally via activation of plasma The modification of protein function and
membrane G proteins (see Chapter 22). regulation of enzyme activity are achieved by
In nervous tissue, calcium is involved in a number of strategies, one of which is cova-
transmission of nerve impulses. Depolarization lent modification by phosphorylation and
of the presynaptic neural membrane results in dephosphorylation. The addition or removal
an influx of calcium through pores known as of one or more phosphate residues induces
voltage-gated calcium channels. The increase conformational changes in proteins which
in intracellular calcium concentration pro- result in changes in the shape and/or size of
motes the fusion of acetylcholine vesicles binding sites or active sites. Examples of
with the presynaptic membrane, resulting in processes where these mechanisms operate
the release of acetylcholine into the synaptic are the transport of sodium and potassium
cleft. across the plasma membrane and control of
Other processes in which calcium is enzyme activity in glycogen metabolism, both
involved are blood clotting, control of gap of which are discussed in Chapter 22.
junction closure between interconnecting Phosphorus is particularly important in the
cells, and cell-to-cell adhesion. metabolism of sugars and polysaccharides.
Abnormalities in calcium metabolism can Simple sugars such as glucose are relatively
occur due to poor dietary intake or to physio- inert unless they are converted to phosphate
logical and metabolic disturbances. In young esters. In glycolysis the first step in the metab-
humans and animals, rickets results from olism of glucose is its conversion to glucose-6-
incomplete mineralization of bones leaving phosphate. All of the intermediate compounds
them soft and unable to support the growing between glucose and pyruvate, the end prod-
body. In adults, abnormal calcium resorption uct of glycolysis, are phosphate esters.
and deposition in mature bones can lead to
conditions such as osteoporosis and osteomala-
8.2.4 MAGNESIUM
cia. In lactating females, particularly high-
yielding dairy cows, the sudden loss of calcium Magnesium is a minor component of bones
in milk at the onset of lactation can lower plas- (0.5-0.7 mg kgI bone) and this accounts for
ma calcium levels dramatically and lead to the approximately 60-70% of the magnesium in the
condition known as milk fever (parturient body. The remainder is found in soft tissues.
hypocalcaemia) which can be fatal if untreated. Almost all of the ATP and ADP found in
cells is complexed to magnesium, as shown in
Figure 8.12. During enzyme-catalysed phos-
8.2.3 PHOSPHORUS
phorylation reactions, magnesium forms a
Phosphorus is widespread in living organisms bridge between the pyrophosphate of ATP
and has functions in more biochemical and the enzyme molecule and lowers the acti-
processes than any other mineral. It is vital to vation energy of the reaction.
energy transduction and oxidation-reduction Other enzymes which require magnesium
reactions in all organisms due to its role in the as a coenzyme include dehydrogenases and
The importance of maintaining the correct
/~;
electrolyte balance in intracellular and extra-
cellular fluids is illustrated by the fact that a
o 0 0 N-t~ major portion of the energy expenditure in
cells is directed towards ion pumping
~P~--P-O-P-O-~CO
1- ,_ 1_ (Chapters 22 and 29).
o 0 0 H H
The movement of sodium across cell mem-
\ I H
\ I
Mg++ OH OH branes is required for the transmission of
nerve impulses and the uptake of nutrients
Figure 8.12 The structure of the magnesium-ATP from the digestive tract and into cells. For
complex. example, the depolarization and repolariza-
tion of neural membranes involves the rapid
enolase. The requirement for magnesium is influx of sodium through voltage-gated sodi-
not absolute and it may be replaced by man- um channels. In the digestive tract sodium,
ganese, another divalent cation. which is present in high concentrations in
Magnesium acts as a counter-ion to nucleic digestive fluids, is co-transported with mono-
acids, which exist as polyanions under physio- saccharides and amino acids across the plasma
logical conditions. Thus, magnesium may membranes of cells lining the lumen of the
influence the transcription of DNA to mRNA small intestine. In contrast chloride, which is
and the translocation of mRNA to protein by required in high concentrations in gastric
stabilizing ribosomal structures and activating secretions, is secreted by the gastric mucosa
the transfer of amino acids from amino acyl (Chapter 28). There are few examples of chlo-
tRNAs to the growing polypeptide. ride acting as a coenzyme and activator of
enzymes, however intestinal amylase is one
enzyme which is activated by chloride.
8.2.5 SODIUM, CHLORIDE AND POTASSIUM
Potassium is required for the transmission
These three elements provide the major elec- of nerve impulses to muscle fibres and in the
trolytes of physiological fluids. Sodium consti- contraction of muscle fibres. It is also an acti-
tutes over 90% of the total cations and chloride vator of a number of enzymes, for example,
in excess of 65% of the anions in blood. hexokinase, pyruvate kinase and fructoki-
Potassium is the major intracellular cation. The nase.
osmotic pressure of extracellular and intracellu-
lar fluid can be markedly changed by excessive
8.2.6 SULPHUR AND IRON
intake and loss of one or more of these ions.
The concentration of Na+, K+ and CI- in a Sulphur occurs as a component of a wide
typical mammalian cell is shown in Table 8.3. range of molecules. The sulphur amino acids
methionine, cystine and cysteine are compo-
Table 8.3 Concentration of sodium, potassium and nents of most proteins. The SH groups of cys-
chloride in intracellular and extracellular fluid in teine stabilize the tertiary and quaternary
mammalian cells
structure of proteins by undergoing oxidation
Ion Intracellular Extracellular to form disulphide bridges with adjacent SH
concentration concentration groups. SH groups also participate in the bind-
(mM) (mM) ing and transformation of substrates and
products on the active sites of enzymes. The
Na+ 5-15 145-150 supply of sulphur amino acids for protein syn-
K+ 140-145 5-10 thesis can be a limiting factor in the growth of
Cl 5-15 110-115
both plants and animals.
Sulphur occurs in a number of other biomol- 8.2.7 OTHER ELEMENTS WITH KNOWN
ecules. It is a component of glycosaminogly- BIOCHEMICAL FUNCTIONS
cans, chondroitin sulphate, dermatan sulphate,
Copper
heparan sulphate and keratan sulphate, which
form the extracellular matrix surrounding cells A number of enzymes require copper. These
in tissues. It is also found in the coenzymes include cytochrome oxidase, tyrosinase, ascor-
lipoic acid and coenzyme A, in acyl carrier pro- bate oxidase and polyphenol oxidase. In addi-
tein and glutathione, and in the vitamins biotin tion, copper is thought to be involved in the
and thiamin. Animal tissue cannot reduce sul- de saturation of fatty acids, and is a component
phate to the SH form found in most proteins of cytoplasmic superoxide dismutase which
and therefore animals are dependent on catalyses the rapid removal of the toxic super-
reduced sulphur compounds in the diet. oxide radical anion 0;-. The active centre of
Rumen microorganisms can, however, use this enzyme contains two copper and two zinc
inorganic sulphur to synthesize amino acids. atoms.
Iron is found in a number of proteins in Copper deficiency blocks the formation of
plants and animals. In many cases it is coor- cross-links essential for structural proteins. For
dinated in the haem prosthetic group, also example, elastin contains many cross-links
called Fe protoporphyrin IX. This gives the between lysine residues in adjacent polypep-
red colour to the oxygen-binding proteins tide chains which render the protein insoluble.
myoglobin and haemoglobin, found in mus- The key copper-containing enzyme required
cle and blood, respectively. It is a component for normal cross-linking is lysyl oxidase.
of the respiratory chain cytochromes. A Copper deficiency in pigs results in failure to
number of non-haem proteins also contain cross-link, and accumulation of a soluble pre-
iron. cursor of elastin has been identified.
In animals transferrin is the major iron- In animal nutrition copper, molybdenum
transport protein found in the blood, and it and sulphur are often considered together
also mediates the uptake of iron by cells. Once because of the interaction between these ele-
in the cell, iron is stored bound to the protein ments in the digestive tract, which can reduce
ferritin (phytoferritin in plants) which con- their availability. This is due to the formation
tains a dense core of ferric hydroxide. of highly insoluble copper thiomolybdates.
A number of iron-sulphur proteins are
components of the mitochondrial electron
Cobalt
transport chain. These proteins contain
iron-sulphur centres with either two or four All the known functions of cobalt are related to
iron atoms bound to an equal number of sul- its role as a component of vitamin B12• These
phur atoms, some of which are components of are discussed above (section 8.1.9). Non-rumi-
the amino acid cysteine. These iron-sulphur nant animals cannot utilize cobalt and require
centres function as electron carriers. There are a supply of preformed vitamin B12 • Because
at least six different iron-sulphur centres in rumen microorganisms can incorporate inor-
the respiratory chain, five of which are known ganic cobalt into the vitamin, these animals are
to occur in the NADH dehydrogenase com- able to take advantage of dietary cobalt.
plex and at least one in the b-c complex
(Chapter 12). In chloroplasts the final transfer
Iodine
of electrons from photosystem I to NADP
reductase is mediated by another iron-sul- In animals iodine is required for the synthesis
phur protein, ferredoxin. Iron-sulphur centres of the thyroid hormones, triiodothyronine (T3)
are also found in a number of enzymes such as and tetraiodothyronine or thyroxine (T4)' For
aconitase and xanthine oxidase. this reason over 90% of the iodine in the body
is concentrated in the thyroid gland. Most the enzyme farnesyl pyrophosphate syn-
dietary iodine is absorbed in the form of iodide thase.
(1-). It is transferred from the circulation to the
thyroid gland by an active transport process Molybdenum
associated with the plasma membrane Na+,
K+-ATPase. In the thyroid gland, iodine is con- A number of molybdenum-dependent
verted to a highly reactive free radical which enzymes have been identified in animals and
rapidly reacts with the phenyl groups of the plants. In animals it is a component of xanthine
tyrosine moieties of thyroglobulin. The iodi- oxidase, aldehyde oxidase and sulphite oxi-
nated protein is stored within the gland and is dase. In desert irrigation water, the concentra-
broken down in lysosomes to release T3 and T4 tion of molybdenum is often high and may lead
when the cells are activated by thyroid-stimu- to a serious risk of secondary copper deficiency.
lating hormone. A characteristic symptom of
iodine deficiency is goitre, an enlargement of Selenium
the thyroid gland. As the gland is unable to Glutathione peroxidase is the only enzyme
synthesize T3 and T4, a hypothyroid state known to contain selenium in animals. This
ensues which has profound effects due to the enzyme is part of the intracellular antioxidant
role of thyroid hormones in the regulation of mechanism and works in concert with superox-
many metabolic processes. ide dismutase, catalase, vitamin E and vitamin
Iodine is found in plant tissues, and marine C. Many of the selenium deficiency symptoms
plants are a particularly rich source. However, observed in animals are related to tissue dam-
iodine does not appear to be an essential ele- age associated with increased levels of lipid
ment in plant nutrition. peroxides. A number of other selenoproteins
have been identified in animal tissues, some of
Manganese which may be selenium transport proteins;
however, no specific functions have been
Manganese is a divalent ion which can sub- assigned to these proteins.
stitute for many of the functions of magne- Unlike animals, higher plants do not contain
sium. In animals, it is an activator of a num- glutathione peroxidase and no other selenium-
ber of enzymes such as hydrolases, kinases, containing enzymes have been identified.
decarboxylases and transferases. This activa-
tion is not always manganese-specific, and
Zinc
other divalent ions may have a similar effect.
However, in the case of glycosyltransferases, Zinc is a component of a large number of
manganese is essential for activation. These enzymes in animal tissues, for example car-
enzymes are required for the synthesis of boxypeptidase, thermolysin, alkaline phos-
polysaccharides, glycoproteins and glyco- phatase, alcohol dehydrogenase, carbonic
lipids. A deficiency of manganese can lead to anhydrase, lactate dehydrogenase and cyto-
bone and joint abnormalities, reflecting the plasmic superoxide dismutase.
role of glycosyltransferases in the synthesis of In both plants and animals, zinc also plays
bone matrix and cartilage. Mitochondria con- an important role in the regulation of DNA and
tain manganese-dependent superoxide dis- RNA metabolism. Regulatory proteins bind to
mutase, and increased lipid peroxidation and these nucleic acids through a number of differ-
membrane dysfunction may be related to a ent recognition sites. Some of these proteins
decrease in the activity of this enzyme in contain a so-called zinc finger, a loop in the
manganese deficiency. Steroid and terpene protein structure which is stabilized by the for-
biosynthesis may also be affected by man- mation of a tetrahedral complex with zinc and
ganese deficiency as it may be required for the amino acids histidine and cysteine.
THE COMPOSITION OF AGRICULTURAL 9
PRODUCTS

9.1.1 Energy storage in animals and plants 133
9.2 The composition of animals 134
9.2.1 Body composition 134
9.2.2 Milk 134
9.3 Plant materials 134
9.4 Principal nutrients in plants and animals 00
9.4.1 Proteins 00
9.4.2 Lipids 00
9.4.3 Carbohydrates 00
9.1 INTRODUCTION the structural elements of the cell and as the

building materials for the vascular system, are
In general the composition of animal tissues taken over by proteins and lipids in animals.
tends to be restricted to a much narrower The amount of carbohydrate in an animal
range, both of substances and of amounts, than body is rarely greater than about 0.2% of body
is found in plants. Animals are to a very great weight; of this, over 90% will be present as
extent composed of water, protein, lipid and glycogen. In some plant tissues the carbohy-
minerals (mainly calcium phosphates). Within drates account for well over 90% of the dry
individual animal tissues there are differences matter.
in the ratios of the principal components but
these are not major. If we exclude the hard
9.1.1 ENERGY STORAGE IN ANIMALS AND
parts of bone then the gross compositions of
PLANTS
the structural, reproductive and digestive tis-
sues from animals are remarkably similar. The difference in choice of energy reservoir in
These similarities persist throughout the whole plants and animals is quite understandable in
of the animal's life cycle. On the other hand, terms of the ways in which plants and animals
plant tissues vary enormously in composition exist. As a store of energy the carbohydrates
both between the different plant tissues and have the advantage of being synthesized and
between the same tissue at different stages in used very easily. Lipids bring with them all
its development. sorts of problems because of the fact that they
The really striking difference between are not easily metabolized in the aqueous
plants and animals lies in their use of carbohy- environment of any biological system. The dis-
drates. Plants use these as one of their main advantage of carbohydrates is that they are
means for storing energy, whereas the long- not as energy-dense as the lipids. One gram of
term energy reserves of animals are almost carbohydrate has a gross energy (the heat of
completely in the form of lipid. The other combustion under standard conditions) of
important roles of carbohydrates in plants, as about 18 MJ kg-l whereas lipids produce
134 The composition of agricultural products
approximately 39 MJ kg-I. In other words, to lipid, protein, minerals and water. Certainly,
store a given amount of energy requires about the animal will contain a small amount of car-
twice the weight of carbohydrate as lipid. To bohydrate, but this is too small even to show
have a heavy fuel storage system is no great on the diagrams. Looking at the two diagrams,
disadvantage to the part of a plant which the striking difference between the young (75
remains rooted in one spot. Animals, on the kg) and the mature (500 kg) animal is in the
other hand, are mobile and carrying extra amounts of fat and water. The mineral fraction
weight would be a distinct handicap. Putting remains quite constant and the protein tends
this into human terms, an average male of 70 to decrease with age, but the fat content
kg body weight probably has 12 kg of fat. To increases more than three-fold. To compensate
store this energy as carbohydrate would add a for the increased fat there is a reduction in the
further 13 kg to his mass without giving any water content (see Chapter 3~}.
compensating advantage. The ratio of the major mineral elements in
Seeds may be regarded as the exception to an animal carcass tends to remain very con-
the rule in plant tissues. They are small, mobile stant throughout life (see Figure 9.2). The six
entities and some, such as rape seed (canola) minerals illustrated form the largest part of
and linseed, take advantage of a high energy/ those present; the trace elements would not
weight ratio by storing a large proportion of even be visible at the scale of this diagram.
their energy reserves as oil.
9.2.2 MILK
9.2 THE COMPOSITION OF ANIMALS Milk is the one agricultural product of animal
origin that contains substantial quantities of
9.2.1 BODY COMPOSITION
carbohydrate, in the form of the disaccharide
In looking at the composition of the animal lactose. Here, the advantage of the high energy/
body, one component that must be ignored is weight ratio is outweighed by the needs of the
the contents of the gut - particularly in herbi- young for an instant and easily metabolized
vores where this may account for a quarter of energy source derived from carbohydrate. The
the animal's overall body weight. The gross milks from different species vary considerably
composition of the carcass (excluding the in composition (Table 9.1); for instance human
digestive tract) of young and mature beef ani- milk has a low protein content, and the milk
mals is shown in Figure 9.1. These proportions from arctic sea-dwelling mammals such as
may be taken as fairly typical for farm live- whales has extremely high levels of fat. The
stock. The only components that are shown are commonest milk in agricultural production
Table 9.1 Composition of milks from various species
Animal Lactose Protein Fat Solids, not fat Calcium

(gkg-I) (gkg-I) (g kg-I) (g kg-I) (g kg-I)
Holstein cow 46 33 35 85 1.2

Jersey cow 49 36 46 90 1.3
Tropical cow (Bos indicus) 46 32 50 85 1.3
Sheep 44 60 75 115 2.0
Goat 42 34 40 87 1.3
Buffalo 49 38 75 90 1.9
Dog 41 83 97 131 3.0
Pig 52 60 83 119 2.7
Horse 63 22 12 85 0.8
Human 68 13 40 90 0.3
Principal nutrients in plants and animals 135
Protein 194 Protein 158
Minerals 28
Lipid 66 Water 519
Lipid 295
(a) (b)
Figure 9.1 Overall composition of the animal body. (a) Calf, body weight 75 kg; (b) steer, body weight
500 kg (figures in g kg-I of carcass).
comes from dairy cattle and the typical com- plant products. It can be seen clearly that they
position is illustrated in Figure 9.3. Milks from form two groups with very high or very low
different breeds of cattle vary slightly in com- water content. In general, the water content has
position, particularly in the fat content, and to be reduced below 150 g kg-I if spoilage is not
there are also changes in response to varia- to occur during storage. Even after successful
tions in the diet and with season. storage, the water content of materials will fluc-
tuate in response to changes in the moisture
content of the environment. For this reason the
9.3 PLANT MATERIALS
composition of most agricultural commodities
The water content of plant materials is even is expressed on a dry matter basis (g kg-I DM or
more variable than that in animals. Figure 9.4 mg kg-I DM, as appropriate). In agricultural
shows the amounts of water in a variety of terms we can divide plant materials arbitrarily
PhosPhorus~8~~~~m~~~~
Calcium 16
Magnesium 0.45 ' ,
Chloride 1.2
Figure 9.2 Mineral composition of a beef steer (g kg-I of carcass).

Fat 36
Protein 32 _...-xI7'7'__
Lactose 46
Minerals 7"'.----4
Water 879
Figure 9.3 The composition of cow's milk (g kg-I of milk).
upon the basis of their chemical composition needs of humans is a supply of the essential
and in most cases this will be related to the use amino acids in approximately the right propor-
that is made of them (Table 9.2). tions. Not surprisingly, the proteins supplied
by animals - meat, fish, eggs and milk - usual-
ly have ratios of amino acids that are closer to
9.4 PRINCIPAL NUTRIENTS IN PLANTS AND
the requirements of humans than are the pro-
ANIMALS
teins from plant sources. Figure 9.5 shows the
pattern of essential amino acids in the proteins
9.4.1 PROTEINS
of fish and those from groundnuts. These are
One of the main purposes of agriculture is the compared with the spectrum that is needed in
provision of food. Amongst the nutritional an animal's diet. It is quite clear that some
1000 rr--Tr- - r r --.,....--Tr--",----,...---rr----n
o Grass Alfalfa Cabbage Potato Beans Grain Straw Hay
Figure 9.4 Typical water content of some plant materials (g kg-I of fresh material; solid bar, water; shaded
bar, dry matter). Note that the water content may vary greatly depending upon storage conditions.
Principal nutrients in plants and animals 137
Table 9.2 Division of plant materials in terms of composition and nutritional use
Water Protein Lipid Sugar Starch Fibre Principal food use

content content content content content content
High protein!low-to-medium fibre

Oil seeds L H H L L M Edible oil extraction
Oil-seed cakes L H L L L M Any livestock
Pulses L H M/H L M/H L Human/non-ruminant
animals
High starcMow-to-medium fibre
Cereal grains L L MIL L H MIL Human or any livestock
Root vegetables H L MIL L H MIL Human or any livestock
High fibre/low-to-medium protein
Grasses H L L MIL M/H H Ruminant/other
herbivore
Forage legumes H M/H L MIL M/H H Ruminant/other
herbivore
High fibre/very low protein
Cereal residues L L L L L H Ruminant/other
e.g. straw herbivore
High moisture/high sugars
Green vegetables H L L H L M Human or any livestock
Fruits H L L H MIL L Human or any livestock
Brassica roots e.g. H L L H L M Human or any livestock
turnip
High fibre/high sugars
Sugarcane H L L H L H Human or any livestock
H = High, M = Medium, L = Low
amino acids such as arginine are over-repre- atures, whereas plant storage lipids are oils (see
sented in the plant protein but that, equally, Tables 4.3 and 4.4).
some such as leucine and methionine are in
short supply. In order to ensure health, a pro-
9.4.3 CARBOHYDRATES
tein source such as groundnut should be sup-
plemented with another that is adequate in Plants contain a huge diversity of carbohy-
leucine and methionine. drates which reflects the variety of purposes to
which they are put. A comparison of two dif-
9.4.2 LIPIDS
ferent carbohydrate-rich sources shows that in
There are characteristic differences between the a grain most of the carbohydrate is either the
lipids of plants and those of terrestrial mam- starch of the grain kernel or the cellulose of the
mals. The most important of these is in the outer protective layers. A green plant such as
degree of unsaturation of the fatty acids. In ani- cabbage has a quite different spectrum of
mal milk fat, most of the fatty acids are either materials, demonstrating that most of the car-
saturated or monounsaturated (C I6, C18:0 and bohydrates are typically those of the cell walls.
CI8 :I ) whereas the fatty acids from a plant such It is perhaps no surprise that there is a dif-
as soyabean are almost all polyunsaturated. In ference between grain and cabbage leaf in
general, lipids from terrestrial animals (espe- their carbohydrates, but there are also varia-
cially the ruminants) are solid at room temper- tions between plants which appear superfi-
g amino acid/kg protein

140 r
120 II (* Amino acid deficient in groundnuts)

II
II
100 IIII
II
*r
II
80 II
II
II
60 :~ IT IT
[I [I II
II 'I II II
40 II II
II
II
II
II
II
'I
'I
'I
* . II
II
II
II
1/
II
II
II
20
rl
1/
II
:; (; rJ J ~I
ij
O~~~~~~U~~~v.~~~~~~.~I~
~ :~~:~II
V
Arg His lieu Leu Lys Met Phe Thre Try Val
[ZZJ Groundnuts • REQUIREMENTS 0 Fish meal

Figure 9.5 Essential amino acids required for growth in non-ruminants compared with the composition
of dietary protein sources.
dally similar. One difference which has con- fructosans in temperate grasses are replaced
sequences in agriculture is the difference by hemicellulose in tropical species. This
between temperate and tropical grasses in means that it is very difficult to make silage
their carbohydrates (Figure 9.6). The grasses (see Chapter 16) with tropical grasses and
contain similar amounts of cellulose, but the they are not as well digested in the ruminant
high levels of easily fermented sugars and gut.
Hemicellulose
Fructosans
Cellulose Cellulose
Temperate grass Tropical grass

Figure 9.6 Comparison of the carbohydrate content of temperate and tropical grasses.
PART TWO
METABOLISM
GLYCOLYSIS 10

10.2 Stage 1 - preparing glucose for splitting 141
into two three-carbon units
10.2.1 Glucose phosphorylation 141
10.2.2 Fructose and its phosphates 143
10.2.3 Splitting of fructose-1,6-bisphosphate 143
10.3 Stage 2 - metabolism of the three-carbon 143
compounds
10.3.1 First oxidation step 143
10.3.2 First energy released in the form of ATP 145
10.3.3 Formation of pyruvate 145
lOA The entry of other sugars 145
1004.1 Fructose 146
1004.2 Galactose 146
1004.3 The entry of glycogen 146
10.1 INTRODUCTION Pyruvate can form the starting material for

a wide range of other pathways. Perhaps
Glycolysis, literally the splitting of sugars,
strangely for a compound that is so important,
occurs in the cytosol of nearly every different
cells never accumulate pyruvate so it is not
cell type of almost all organisms, from bacteria
found in large quantities.
to higher animals. The enzymes are soluble
rather than being bound to membranes. It has
two main roles: 10.2 STAGE 1- PREPARING GLUCOSE FOR
SPLITTING INTO TWO THREE-CARBON
• oxidizing sugars and, in the process, pro-
UNITS
ducing energy for use in other parts of the
cell; The first few steps of the pathway involve the
• breaking sugars into smaller molecules with production of a sugar molecule which has
two, three or four carbon atoms that can be phosphate groups attached to it, as illustrated
used as starting materials for the synthesis in Figure 10.1.
of other important chemical compounds.
The pathway consists of two sections.
10.2.1 GLUCOSE PHOSPHORYLATION
Firstly, a molecule of glucose (or of any hex-
ose) is converted to fructose-1,6-bisphosphate Glucose is taken into cells by active transport
(fructose that bears a phosphate group at each and is then phosphorylated at the 6 position.
end). This six-carbon compound is broken into The phosphate group comes from ATP and the
two three-carbon molecules. The second part reaction is catalysed by one of two enzymes,
of the pathway converts both of these three- hexokinase or glucokinase. These belong to a
carbon residues to pyruvate (pyruvic acid) - large group of enzymes known as the kinases,
still with three carbons. which phosphorylate compounds using ATP.
142 Glycolysis
~
C~O:
Glucose
OH
HO OH
OH
v-;:.o~::se / glucokinase
~ADP
HO
J,P®> OH
Gluco.e-5-p'o.p'''.
Jt :SPhOgIUCOSe isomerase
®O~O~H20H
Fructose-6-phosphate
~ OH
k:; ATP
Phosphofructokinase
ADP
®OCH 2
o OH
° CH20®
F,ucto•• ',6-.,.p'.,p""
~~O®
A HC
I
=0
c=o HCOH
I I
CH 20H CH 20®
dlhydroxyacetone
Glyceraldehyde-3-phosphate
phosphate
Figure 10.1 The first stages of glycolysis.
Like other kinases, these enzymes have a glucose + ATP -+

requirement for Mg2+ ions; other ions, particu- glucose-6-phosphate + ADP
larly Mn2 +, will act instead but they are proba-
bly not the normal intracellular cofactors. Hexokinase is found in muscle and glucok-
Both enzymes catalyse the conversion of inase in liver. The KM of hexokinase for glucose
glucose to glucose-6-phosphate. is about 0.1 mM and that for glucokinase 10
Stage 2 - metabolism of the three-carbon compounds 143
mM. The concentration of glucose in the blood 10.2.3 SPLITTING OF FRUCTOSE-1,6-
varies between about 4 and 8 mM as a result of BISPHOSPHATE
ingestion of carbohydrates. Both of these val-
Fructose-1,6-bisphosphate is broken into two
ues are well above the KM for hexokinase, so
molecules, each with three carbon atoms and a
that this enzyme is saturated all of the time - it
phosphate group. This reaction is catalysed by
is working at maximum rate and the velocity
the enzyme aldolase, which is the last reaction
of formation of glucose-6-phosphate in muscle
of the sequence shown in Figure 10.1. The free
is independent of ingestion of carbohydrates.
energy change of the reaction is small and
However the KM of glucokinase is similar to
thus it can be reversed.
the glucose concentration in blood, and
changes in blood glucose levels therefore dra- 10.3 STAGE 2 - METABOLISM OF THE THREE-
matically change the rate of glucose-6-phos- CARBON COMPOUNDS
phate formation and hence glycogen produc-
tion in the liver. Formation of glycogen there- These are phosphorylated versions of the two
fore increases substantially after a meal when three-carbon sugars (Chapter 3). They are
there is plenty of glucose in the blood. structural isomers of one another. The two
Whichever enzyme is involved, the overall different products of the breakdown of fruc-
standard free energy change of -17 kJ mole-1 tose-1,6-bisphosphate can be interconverted
is quite large; the reaction will thus proceed by the enzyme triosephosphate isomerase
easily and spontaneously in the direction of (see Figure 10.2). Like many reactions catal-
the formation of glucose-6-phosphate, but it ysed by isomerases, the reaction is quite
is practically irreversible under biological freely reversible. Therefore, each molecule of
conditions. glucose effectively gives two molecules of
glyceraldehyde-3-phosphate.
10.2.2 FRUCTOSE AND ITS PHOSPHATES
10.3.1 FIRST OXIDATION STEP
Glucose-6-phosphate then has to be trans-
formed into its structural isomer, fructose 6- If we are following the products formed from
phosphate, which is accomplished by the a molecule of glucose, from now on we must
enzyme glucose phosphate isomerase. In remember to double the throughput of the
order to change the aldehyde group of glu- pathways that follow.
cose into the ketone of fructose the enzyme Up to this point the pathway has merely
must convert the sugars into the straight- split the hexose sugar but no energy has been
chain form. extracted. The cell has had to use two mole-
The second phosphate group is then intro- cules of ATP for every molecule of glucose
duced at the opposite end (carbon 1) of the entering the pathway. The next steps repre-
fructose molecule. The product is fructose-1,6- sent the first points at which some energy (in
bisphosphate. The enzyme is phosphofructo- the form of ATP) is returned to the cell.
kinase (PFK). Like the previous kinase reaction, Glyceraldehyde-3-phosphate undergoes two
the free energy change is large and negative changes simultaneously under the influence
(about -14 kJ mole-1). Again, the reaction will of the enzyme glyceraldehyde-3-phosphate
proceed easily in the direction of the forma- dehydrogenase. As its name implies, this
tion of fructose-1,6-bisphosphate and is effec- enzyme takes hydrogen away from its sub-
tively irreversible. The irreversibility of this strate; it also adds another phosphate group to
step forms the basis of the main mechanism form l,3-bisphosphoglycerate. This time the
regulating the flow through the pathway (see phosphorylation proceeds without using ATP,
Chapter 22). and inorganic phosphate is the donor. The
144 Glycolysis
Fructose 1 ,6- dlhydroxyacatona

blsphosphate phoaphata
\t Tri~sephosPhate
~\ Isomerase
HC = 0
bOH Glycaraldahyda
HI 3-phHphata
CH 2 0®
PI + NAD+
Glyceraldehyde-3-phosphate ~
dehydrogenase
NADH + H+
O~
~C-O®
I 1,3-blaphoaphoglycarata
HCOH
CH 2
I
0®
Phosphoglycerate ~ ADP
kinase
o ATP
~C-OH
I
HCOH 3-phoaphoglycarata
I
l'
CHP@
Phosphoglycero
mutase ~
o~
~C-OH
I
HC-O@ 2-phoaphoglycarata
I
CHpH
Enolase
O~ O~
C-OH
Pyruvate
kinase
~C-OH
I
=0 I
T\
Pyruvate C
I
c-o@
II
CHs C Phoaphoanolpyruvata
H/ 'H
ATP ADP
Figure 10.2 The final stages of glycolysis.

The entry of other sugars 145
hydrogen is removed by the coenzyme NAD+. molecule of ATP. The 'balance sheet' (Table
For this process to continue, there must be a 10.1) for phosphate groups is now equal.
constant renewal of the supply of NAD+; this
can be achieved in a number of ways depend-
10.3.3 FORMATION OF PYRUVATE
ing upon the presence or absence of oxygen.
The final part of the glycolytic pathway is the
formation of pyruvate from 3-phosphoglycer-
10.3.2 FIRST ENERGY RELEASED IN THE FORM
ate. The sequence of reactions starts with the
OFATP
rearrangement of 3-phosphoglycerate to give
The next step yields some profit in terms of 2-phosphoglycerate. The reaction is catalysed
energy: one of the phosphate groups is by the enzyme phosphoglyceromutase. The
removed and is used to produce ATP from free energy change in the reaction is quite
ADP. Phosphoglycerate kinase transfers the small, so it is freely reversible.
phosphate group from the 1 carbon of the sub- The next step, which is also freely
strate, 1,3-bisphosphoglycerate, to ADP.1t thus reversible, involves the removal of the hydrox-
has a similar (but reverse) action to the hexoki- yl group on the carbon 3 and a hydrogen at
nase that added phosphate groups to glucose. carbon 2 to form a double bond, producing
For this reason it is also a kinase enzyme, but water and phosphoenolpyruvate This is an
one which is working in the opposite direction. important intermediate in its own right, par-
The free energy change for the reaction is quite ticularly in reactions that synthesize glucose.
small and thus the reaction is reversible - its Pyruvate is produced from phospho-
very name, phosphoglycerate kinase, implies enolpyruvate by the transfer of the phosphate
that it works in the opposite direction to the group to ADP. The reaction, which is catalysed
one we are considering at the moment. The by pyruvate kinase, has a large negative free
product of this step, 3-phosphoglycerate, is one energy change, rendering it irreversible.
point at which other pathways can interact The overall balance sheet (Table 10.2) now
with glycolysis; this intermediate is the point of shows that more energy has been extracted in
departure for the synthesis and breakdown of the form of ATP than had to be put in at the
glycerol. start.
Each molecule of glucose used in this path-
way required two molecules of ATP to get as
10.4 THE ENTRY OF OTHER SUGARS
far as fructose 1,6-bisphosphate. But each
molecule of glucose gave two molecules of 1,3 Although glucose is important, it is not the
bisphosphoglycerate which each gave one only sugar that is encountered in animals and
Table 10.1 Energy balance for the production of 3-phosphoglycer-

ate from glucose
Entering pathway Leaving pathway
One molecule of glucose Two molecules of 3-phosphoglycerate

Two molecules of ATP Two molecules of ATP
Two molecules of NAD+ Two molecules of NADH
146 Glycolysis
Table 10.2 Energy balance for the production of pyruvate
from glucose
Entering pathway Leaving pathway
One molecule of glucose Two molecules of pyruvate

Two molecules of ATP Four molecules of ATP
Two molecules of NAD+ Two molecules of NADH
plants. As examples, many plants both pro- (changing UDP for phosphate) leading to the
duce and use large quantities of fructose. The production of UDP-galactose and glucose-I-
suckling mammal obtains half of its sugar from phosphate (Figure 10.3):
the galactose of milk lactose.
UDP-glucose + galactose-I-phosphate ~
UDP-galactose + glucose-I-phosphate
10.4.1 FRUCTOSE
The glucose-I-phosphate can enter the
It is not difficult to see how fructose enters gly- glycolytic pathway after its conversion to
colysis, it merely needs to be phosphorylated glucose-6-phosphate by the enzyme phos-
to fructose-6-phosphate to enter the pathway. phoglucomutase.
Many plants and animals have a specific To complete the cycle of reactions, UDP-
enzyme, fructokinase, which catalyses the glucose can be regenerated from UDP-galac-
reaction: tose by the enzyme UDP-galactose-4-
epimerase.
fructose + ATP --->
fructose-6-phosphate + ADP UDP-galactose ~ UDP-glucose
In animals, hexokinase is able to phospho- The overall effect is that one molecule of
rylate fructose as well as glucose. ATP is used to produce one molecule of glu-
cose-I-phosphate from a molecule of galac-
tose.
10.4.2 GALACTOSE
The entry of galactose is not as simple as that of
10.4.3 THE ENTRY OF GLYCOGEN
fructose: it has to be converted into glucose-6-
phosphate before it can be used. During many The pathway for the breakdown of glycogen is
sugar transformations the sugar molecule has designed to provide a rapidly available source
to be attached to a nucleotide, this pattern is of energy. Under the influence of the enzyme
seen in the synthesis of disaccharides such as glycogen phosphorylase, glucose units are
lactose and in the formation of starch (Chapter removed from the non-reducing ends of the
18). The nucleotide used in these transforma- chains. The sugars removed are therefore those
tions is uridine diphosphate (UDP) although with a free hydroxyl group at the 4 position.
the coupling is not a simple one. The process is phosphorolysis rather than
Galactose is first phosphorylated directly at hydrolysis, involving the attack of a phosphate
the 1 position: group on the glycosidic link to yield a short-
ened version of the glycogen and a molecule of
galactose + ATP ~
glucose-I-phosphate. This can then be trans-
galactose-I-phosphate + ADP
formed into glucose-6-phosphate ready for
In the next step there is an exchange in acti- entry into the glycolytic pathway (phospho-
vating groups between glucose and galactose glucomutase). In the case of liver glycogen, the
The entry of other sugars 147
Glucose
Galactose 1-phosphate
AlP
ADP Galactose UDP-Glucose
1-phosphate
Glucose UDP-Galactose
1-phosphate
Figure 10.3 Conversion of galactose to glucose-I-phosphate prior to its metabolism by the glycolytic
pathway.
glucose-6-phosphate can be dephosphorylated utilization. If glycogen were a simple, linear,

to yield free glucose, to be used in supplying unbranched molecule then only one glucose
the peripheral tissues of the body. group per molecule would be available to
glycogen phosphorylase at anyone time. The
(glycogen)n + Pi --->
branched structure means that a large number
(glycogen)n_l + glucose-I-phosphate
of non-reducing glucose units are always
The branched structure of glycogen brings available.
with it a great advantage in promoting rapid
THE TRICARBOXYLIC ACID CYCLE 11

11.2 The reactions of the TCA cycle 149
11.2.1 Production of acetyl-CoA 149
11.2.2 Reactions leading to the production 150
of CO 2
11.2.3 Reactions leading back to the formation 150
oxaloacetate
11.2.4 Overall reactions of the tricarboxylic 153
acid cycle
11.3 Links with other metabolic pathways 153
11.4 Replenishment of TCA cycle 155
intermediates
11.5 Conversion of propionate to glucose 155
via the TCA cycle
11.6 Regulation of the TCA cycle 155
11.7 The glyoxylate cycle 156
11.1 INTRODUCTION intermediary metabolism. A glance at a map of

metabolic pathways will quickly reveal that
Like all organic compounds, glucose will burn
the TCA cycle has many inputs and serves to
in air to produce carbon dioxide and heat. This
oxidize not only pyruvate, but also the prod-
oxidation process has been harnessed by liv-
ucts of amino acid and fatty acid catabolism.
ing organisms not to produce heat, but to trap
chemical energy in the form of ATP which can Furthermore, it provides intermediates for
be used for a variety of cellular functions. many biosynthetic pathways such as amino
acid synthesis, fatty acid synthesis and the
We have already seen that the first stage in
production of glucose via gluconeogenesis.
the oxidation of glucose, glycolysis, can pro-
duce a small quantity of useful energy. For Although it is generally agreed that in plants
and animals the reactions of the cycle are the
each molecule of glucose converted to pyru-
same, regulation of the pathway differs.
vate, two molecules of ATP are produced
directly, together with two molecules of
NADH which can be used to produce ATP in 11.2 THE REACTIONS OF THE TCA CYCLE
the electron transport chain (see Chapter 12).
11.2.1 PRODUCTION OF ACETYL-COA
Under aerobic conditions much more ener-
gy can be extracted from glucose if the pyru- In theory a cycle has no beginning or end, but
vate produced by glycolysis is completely oxi- it is necessary to consider the reactions of the
dized to carbon dioxide and water. This is TCA cycle in sequence, and it is accepted con-
achieved by the tricarboxylic acid (TCA) cycle, vention to describe the cycle from the point at
also known as the citric acid or Krebs cycle. which it is fed by the glycolytic pathway. The
This cycle can be considered as the hub of production of pyruvate by glycolysis occurs in
150 The tricarboxylic acid cycle
the cytoplasm. The metabolism of pyruvate this reaction effectively irreversible (Figure
via the TCA cycle occurs in the mitochondrial 11.2).
matrix, thus pyruvate must be transported Citrate undergoes a rearrangement to form
into the mitochondrion before being further isocitrate via cis-aconitate. The reaction is
oxidized. The mitochondrion is bounded by a catalysed by the enzyme aconitase (Figure
double membrane. The outer membrane is rel- 11.3). cis-Aconitate remains bound to the
atively permeable; however, the inner mem- enzyme and is, therefore, found in only very
brane is not and pyruvate must cross this low concentrations in the cell.
membrane via a specific carrier system, the The conversion of isocitrate to a-ketoglu-
pyruvate translocase. tarate is the first of two oxidative decarboxyla-
Pyruvate does not feed directly into the tion reactions of the TCA cycle. Isocitrate loses
TCA cycle but must first be converted to acetyl a carbon atom as CO 2 and is oxidized by the
coenzyme A, usually referred to as acetyl-CoA transfer of hydrogen to NAD+ or NADP+ to
(Figure 11.1). form NADH or NADPH. This reaction is cata-
This conversion is catalysed by pyruvate lysed by isocitrate dehydrogenase (Figure
dehydrogenase (PDH). This enzyme complex 11.4). Two forms of this enzyme occur in most
catalyses the irreversible oxidative decarboxy- microorganisms, plants and animals: (a)
lation of pyruvate to yield acetyl-CoA, CO 2 and NAD+ -dependent isocitrate dehydrogenase,
NADH. It consists of three different enzymes: and (b) NADP+ -dependent isocitrate dehydro-
pyruvate dehydrogenase, containing thiamin genase. The NAD+ -dependent enzyme occurs
pyrophosphate as a coenzyme; dihydrolipoyl only in mitochondria, whereas the NADP+-
transacetylase; and dihydrolipoyl dehydroge- dependent enzyme is found in both the mito-
nase, a flavoprotein containing FAD. The PDH chondrial matrix and the cytoplasm. It is gen-
complex controls the flux of carbon into the erally accepted that the NAD+ -dependent
TCA cycle from glucose. enzyme is the major catalyst of isocitrate oxi-
dation in the TeA cycle whereas the NADP+-
dependent enzyme is important in the synthe-
11.2.2 REACTIONS LEADING TO THE
sis of NADPH in the cytoplasm (see Chapter
PRODUCTION OF CO2
19 on fatty acid synthesis).
The first reaction per se of the TCA cycle is the A second carbon atom is lost as CO 2 in the
condensation of the two-carbon compound, next reaction of the cycle in which a-ketoglu-
acetyl-CoA, with the four-carbon compound, tarate undergoes oxidative decarboxylation to
oxaloacetate, to form a new six-carbon com- produce succinyl-CoA (Figure 11.5). This reac-
pound, citrate. This reaction is catalysed by tion is catalysed by a-ketoglutarate dehydro-
the enzyme citrate synthase. The hydrolysis genase, which is similar in both structure and
of the thioester bond in acetyl-CoA during mechanism to the pyruvate dehydrogenase
the condensation is highly exergonic, making complex. It consists of three enzymes, a-
ketoglutarate dehydrogenase, dihydrolipoyl
HSCoA
succinyltransferase and dihydrolipoyl dehy-
drogenase.
oII
CH 3-C-COOH
11.2.3 REACTIONS LEADING BACK TO THE
Pyruvate Acetyl-CoA FORMATION OF OXALOACETATE
NAD NADH + H+ Reference to Figure 11.6 shows that at this

Figure 11.1 Conversion of pyruvate to acetyl-CoA point in the cycle two molecules of CO 2 have
by the pyruvate dehydrogenase complex. been produced. The cycle started with the
The reactions of the TeA cycle 151
Citrate
O=C-COOH synthase
I
+ CH 2-COOH
Acetyl-CoA Oxaloacetate Citrate
Figure 11.2 The condensation of acetyl-CoA with oxaloacetate to yield citrate.
CH 2-COOH CH 2-COOH CH 2-COOH

I I I
HO-C-COOH .:;;==~"
'4
C-COOH .:;;==~" CH-COOH
" '4 I
CH-COOH HO-CH-COOH
Citrate cis-Aconitate Isocitrate
Figure 11.3 The conversion of citrate to isocitrate via cis-aconitate.
input of the two-carbon compound acetyl- is reduced to FADHz during the course of the
CoA. Thus, there has been a net oxidation of reaction (Figure 11.8). The enzyme is located
acetyl-CoA to carbon dioxide. The remaining on the surface of the inner mitochondrial
reactions of the cycle not only bring about the membrane where it interfaces with the mito-
regeneration of oxaloacetate but also produce chondrial electron transport chain, allowing
the high-energy compounds GTP (ATP in electrons from FADHz to pass along the chain
plants and bacteria), FADHz and NADH which to oxygen, with the consequent synthesis of
can subsequently be used to synthesize ATP. ATP (Chapter 12).
GTP is synthesized by the hydrolysis of the The next reaction of the cycle is the addition
energy-rich thioester bond in succinyl-CoA to of a molecule of water across the trans double
yield succinate. The reaction is catalysed by bond of fumarate to produce L-malate. The
the enzyme succinyl-CoA synthetase, some- enzyme responsible for this conversion is
times also referred to as succinate thiokinase fumarase (Figure 11.9). This type of stereospe-
(Figure 11.7). cific addition of water occurs elsewhere in
Succinate is subsequently oxidized to metabolic pathways. For example, the enolase
fumarate by the action of succinate dehydro- reaction of fatty acid oxidation in which the
genase, a flavoprotein containing FAD which addition of water across the trans double bond
of the enoyl-CoA intermediate produces an L-
3-hydroxyacyl-CoA intermediate. Fumarase is
9H2-COOH 9H2-COOH
specific for the trans double bond of fumarate
CH-COOH
I 9H 2 and will not act on the cis isomer of fumarate,
HO-CH-COOH O=CH-COOH known as maleate.
CO2 The final reaction of the cycle is the oxida-
/socitrate tion of malate to form oxaloacetate and
Isocitrate a-Ketoglutarate
dehydrogenase NADH. This reaction is catalysed by NAD+-
Figure 11.4 The conversion of isocitrate to a-keto- dependent malate dehydrogenase (Figure
glutarate by isocitrate dehydrogenase. 11.10). The reaction is freely reversible, but
NAO+ NAOH + H+
CH 2-COOH CH 2-COOH
I I
CH 2 CH 2
I I
O=CH-COOH O=C-SCoA
a-Ketoglutarate crKetoglutarate Succinyl-CoA

dehydrogenase
Figure 11.5 The conversion of a-ketoglutarate to succinyl-CoA by a-ketoglutarate dehydrogenase.
because oxaloacetate is rapidly utilized for the found in the cytoplasm where it catalyses the
synthesis of citrate, the reaction is 'pulled' in formation of malate from oxaloacetate. Also
the direction of oxaloacetate synthesis. NAD+- found in the cytoplasm is an NADP+ -depen-
dependent malate dehydrogenase is also dent malate dehydrogenase which catalyses
Pyruvate
CH,~
Oxaloacetate ~ ~I',
~ HC).C.(XX)H . Citrate
:~+~~ =\
I
~ lsocitrate
::-t-
~ r~~-"
~K~+w
=:= '7:::::oc:
Fumarate ~ fHtCOOH
~ ?Hz a-l<etoglutarate
F~'~=--
__ f 'r"""'" .~ I... ~ \\ CO,
.~ ~
"-77 '\
FAD .. _synthetase .. . NADH + H+
Succinate Succiny\-CoA
HSCoA GTP GOP + Pi
Figure 11.6 The reactions of the TCA cycle.
Links with other metabolic pathways 153
H2 O
""-
CH-COOH HO-CH-COOH
CH 2-COOH
I
CH2-COOH
I CH
II
'4
. I
CH2
9H
I
9H 2
COOH
2 COOH
Fumarase
COOH
O=C-SCoA Succinyl-CoA
synthetase
Succinyl-CoA Succinate
Fumarate Malate
Figure 11.7 The conversion of succinyl-CoA to suc-
cinate by succinate synthetase. Figure 11.9 The conversion of fumarate to malate.
the oxidative decarboxylation of malate to processing of the NADH and FADH2 which
pyruvate. To distinguish the two forms of the have been synthesized. These must be re-oxi-
enzyme, the NADP+ -dependent form is often dized to NAD+ and FAD if the cycle is to con-
referred to as the malic enzyme. tinue to function and it is in this oxidation, by
a process called oxidative phosphorylation,
11.2.4 OVERALL REACTIONS OF THE that useful quantities of ATP are produced.
TRICARBOXYLIC ACID CYCLE The complete oxidation of one mole of glucose
via glycolysis and the TCA cycle leads to the
In the absence of inputs to the cycle other than production of 38 moles of ATP (Table 11.1).
acetyl-CoA, or of the removal of intermediates
for biosynthetic purposes, the net effect of the
TCA cycle can be summarized as shown 11.3 LINKS WITH OTHER METABOLIC
below: PATHWAYS
CH3COSCoA + GDP + Pi + 3NAD+ FAD + Under most circumstances in both plants and
2HzO -> 2C02 + GTP + 3NADH + FADH2 + animals the TCA cycle is fed by pyruvate
2H+ + CoASH derived from glucose, but it has many links
with other metabolic pathways, and many
Thus, acetyl-CoA is oxidized to two mole- catabolic processes produce end products
cules of CO2, Oxaloacetate does not appear in which can feed directly into the cycle. In addi-
the equation as, although it condenses with tion, intermediates of the cycle can be
acetyl-CoA to produce citrate, it is regenerated 'siphoned off for use as the starting point of
in the last reaction of the cycle. The production numerous biosynthetic pathways.
of GTP by the cycle leads to the production of Quantitatively, the most important input
ATP through the action of the enzyme nucleo- into the TCA cycle is pyruvate produced by
side diphosphate kinase and thus adds to the the glycolytic pathway. However, fatty acid
energy stores of the cell (Figure 11.11). oxidation can also make an important contri-
The largest contribution of the cycle to bution to the supply of acetyl-CoA required
energy production comes from the further
FAD FADH2
9H2-COOH
9H 2
\.) ..
CH-COOH
I
CH
I
HO-CH-COOH
I
9H2
O=C-COOH
I
9H 2
COOH Succinate COOH COOH Malate COOH
dehydrogenase dehydrogenase
Succinate Fumarate Malate Oxaloacetate
Figure 11.8 The conversion of succinate to Figure 11.10 The conversion of malate to oxalo-
fumarate. acetate.
nism allows the conversion of storage lipid to
GTP+ADP ..
Mtj+
.. GDP+ATP sucrose which is more readily transported
around the plant.
Figure 11.11 The conversion of GTP to ATP. Amino acids can also supply fuel for the
TCA cycle and feed into the cycle at various
for TCA cycle function. This is particularly points. Thus, for example, glutamate and
important in animals that are in negative ener- aspartate undergo transamination to produce
gy balance. In this situation glucose oxidation a-ketoglutarate and oxaloacetate, respectively;
is greatly reduced in response to a fall in blood valine and isoleucine are metabolized to suc-
glucose concentrations. When this occurs, cinyl-CoA; and leucine and lysine produce
fatty acids are mobilized from adipose tissue acetyl-CoA. The relationship between amino
depots and undergo f3-oxidation in mitochon- acids and the TCA cycle is discussed in
dria to form acetyl-CoA which enters the TCA Chapter 14. In plants, it is unlikely that much
cycle (see Chapter 13). This has important ben- amino acid is metabolized via the TCA cycle.
efits because it allows those tissues which can Most evidence suggests that amino acids
readily oxidize fatty acids to meet their ATP released by protein breakdown are transport-
requirements, while sparing glucose for those ed to other tissue sites and stored or used
tissues that are essentially dependent on glu- directly for protein synthesis.
cose as an energy source (e.g. the brain and The main outflow from the TCA cycle in ani-
nervous tissue) or those tissues that have a mals is directed towards the synthesis of fatty
specific demand for glucose for biosynthetic acids, amino acids and glucose. In non-rumi-
processes (e.g. lactose synthesis in mammary nant animals, the main source of the acetyl-
tissue). In plants, there is little evidence that CoA required for fatty acid synthesis is glucose.
storage lipids are oxidized via the TCA cycle in This undergoes partial oxidation via the gly-
significant quantities. Fatty acid oxidation in colytic pathway to produce pyruvate, which is
plants occurs mainly in the glyoxysomes not in then converted to mitochondrial acetyl-CoA.
the mitochondria, and the acetyl-CoA pro- Translocation of acetyl-CoA to the cytoplasm
duced enters the glyoxylate cycle rather than occurs via citrate which is split into its two
the TCA cycle (see section 11.6). This mecha- component parts, acetyl-CoA and oxaioacetate,
Table 11.1 ATP production from the oxidation of glucose via glycolYSis and the TCA cycle
Substrate Cofactor ATP ATP

produced consumed produced
glucose - glucose-6-phosphate 1
fructose-6-phosphate - fructose-l,6-bisphosphate 1
2 x glyceraldehyde-3-phosphate - 1,3-bisphosphoglycerate 2NADH 6
2 x 1,3-bisphosphoglycerate - 3-phosphoglycerate 2
2 x phosphoenolpyruvate - pyruvate 2
2 x pyruvate - acetyl-CoA 2NADH 6
2 x isocitrate - a-ketoglutarate 2NADH 6
2 x a-ketoglutarate - succinyl-CoA 2NADH 6
2 x succinyl-CoA - succinate 2GTP 2
2 x succinate - fumarate 2FADH2 4
2 x malate - oxaloacetate 2NADH 6
2 40
Net synthesis 38
Regulation of the TCA cycle 155
by the cytoplasmic enzyme ATP-citrate lyase. cific points. In animals the most important of
The acetyl-CoA can then be utilized for fatty these reactions is the carboxylation of pyru-
acid synthesis (see Figure 19.1). In plants, the vate to produce oxaloacetate. This reaction is
importance of TCA cycle intermediates in fatty catalysed by the allosteric enzyme pyruvate
acid and isoprenoid synthesis is less clear. It carboxylase. The main activator of this enzyme
has been suggested that mitochondrial acetyl- is acetyl-CoA, and when the intramitochon-
CoA is hydrolysed by acetyl-CoA hydrolase to drial concentration of acetyl-CoA is low the
free acetate, which diffuses from the mitochon- enzyme is virtually inactive. However, if the
dria to chloroplasts where it can be reactivated flux through the TCA cycle slows and the con-
and used for fatty acid synthesis. However, centration of acetyl-CoA increases, pyruvate
evidence for the existence of free acetate in carboxylase is activated, making more oxaloac-
plant cells is not convincing, and the ATP-cit- etate available for condensation with acetyl-
rate lyase route may be the more important CoA and thereby increasing the flux through
source of chloroplast acetyl-CoA. the cycle. A number of other reactions also
TCA cycle intermediates can be used for the replenish the supplies of TCA cycle intermedi-
synthesis of many amino acids, for example ates. These include conversion of phospho-
the direct transamination of pyruvate, enolpyruvate to oxaloacetate by phospho-
oxaloacetate and a-ketoglutarate yields ala- enolpyruvate carboxykinase in animals and by
nine, aspartate and glutamate, respectively. phosphoenolpyruvate carboxylase in plants.
Glutamate derived from the TCA cycle can An anaplerotic reaction common to both
then be converted to arginine, proline and glu- plants and animals is the conversion of pyru-
tamine. Oxaloacetate can also be converted via vate to malate by the malic enzyme. These
phosphoenolpyruvate to serine, glycine, cys- reactions are shown in Figure 11.12.
teine, phenylalanine, tyrosine and trypto-
phan. The links between the TCA cycle and
11.5 CONVERSION OF PROPIONATE TO
the synthesis of amino acids are discussed in
GLUCOSE VIA THE TCA CYCLE
Chapter 20.
TCA cycle intermediates can be directed Propionate is a three-carbon compound which
towards the synthesis of carbohydrate, mainly can be converted to glucose. This conversion is
glucose. This is achieved by conversion of particularly important in ruminant animals
oxaloacetate to phosphoenolpyruvate which where, because of the fermentation of dietary
feeds into the gluconeogenic pathway (see carbohydrate in the rumen, there is little or no
Chapter 18). supply of glucose to the animal directly from
the digestive tract. The conversion of propi-
onate to glucose is discussed in more detail in
11.4 REPLENISHMENT OF TCA CYCLE
Chapter 18.
INTERMEDIATES
When the TCA cycle serves as a source of pre-
11.6 REGULATION OF THE TCA CYCLE
cursors for biosynthetic reactions the concen-
tration of cycle intermediates is depleted. Because of its central position in the pathways
Unless the supply of these intermediates is of intermediary metabolism, the TCA cycle is
replenished, the flux of carbon through the subject to stringent regulation at a number of
TCA cycle slows. However, under most condi- points. The main entry point into the cycle is
tions the concentration of cycle intermediates via acetyl-CoA which can be supplied from
remains relatively constant. This balance is glucose via pyruvate or by fatty acid oxidation.
achieved by anaplerotic reactions which feed The conversion of pyruvate to either acetyl-
intermediates back into the TCA cycle at spe- CoA or oxaloacetate is controlled by the
pyruvate carboxylase
Pyruvate + HC0 3+ + ATP ..;;;.r=......= ..................~t oxaloacetate + AOP + Pi
phosphoenolpyruvate
carboxykinase
Phosphoenolpyruvate + CO2 + GOP • • oxaloacetate + GTP
pyruvate carboxylase
Phosphoenolpyruvate + He03- '4 b oxaloacetate + Pi
malic enzyme
Pyruvate + HC03- + NAOPH + H+ • • malate + NAOP+
Figure 11_12 Reactions which replenish the supply of TCA cycle intermediates.
allosteric regulation of the PDH complex or reduced by phosphorylation which occurs in

pyruvate carboxylase, respectively. The PDH response to increased concentrations of
complex is activated by its substrate, pyruvate. NADH and acetyl-CoA.
It is inhibited by its product, acetyl-CoA, and The three enzymes in the TCA cycle which
by long-chain fatty acyl-CoAs. This pattern of catalyse irreversible reactions, citrate synthase,
inhibition provides a mechanism which limits isocitrate dehydrogenase and ex-ketoglutarate
the use of glucose for ATP synthesis when dehydrogenase, also regulate the activity of
fatty acids are available. Pyruvate carboxylase, the cycle. Their activity is modified by changes
on the other hand, is activated by acetyl-CoA. in the energy status of the cell which are
The opposing effects of acetyl-CoA on these reflected in the ratios of the concentrations of
two enzymes have a number of benefits. For ATP/ADP and NADH/NAD+. In general, high
example, when the TCA cycle functions in an concentrations of end product, ATP and/or
aerobic tissue, primarily to supply ATP, there NADH, have inhibitory effects.
is little requirement for additional input of
oxaloacetate into the cycle, and the acetyl-CoA
11.7 THE GLYOXYLATE CYCLE
concentration in the mitochondria remains rel-
atively low due to its constant oxidation. If, Located in special subcellular organelles, the
however, cycle intermediates are removed to glyoxysomes, this pathway occurs in most bac-
supply biosynthetic pathways, the supply of teria, protozoa, fungi, algae and higher plants.
oxaloacetate will be reduced so that acetyl- The cycle provides a means by which sugars
CoA may not be utilized as rapidly as it is pro- and other important cellular metabolites can
duced. This can result in an increase in the be synthesized from two-carbon compounds
mitochondrial concentration of acetyl-CoA. such as acetate and ethanol.
The higher acetyl-CoA concentration reduces The conversions of phosphoenolpyruvate
the activity of the PDH complex and increases to pyruvate, and of pyruvate to acetyl-CoA,
the activity of pyruvate carboxylase, thereby have large negative free energy changes and
restoring oxaloacetate availability. are therefore effectively irreversible. Thus,
In addition to allosteric modification, the acetyl-CoA derived, for example, from fatty
PDH complex may also be regulated by phos- acid oxidation cannot be converted directly to
phorylation/dephosphorylation. Its activity is pyruvate and on to glucose. Instead, glucose
The glyoxylate cycle 157
synthesis is achieved from a variety of gluco- produced in these reactions, the overall effect
genic intermediary metabolites which feed of the glyoxylate cycle is the utilization of two
into the gluconeogenic pathway at a number molecules of acetyl-CoA for the net synthesis
of different points. The most important of of oxaloacetate. This oxaloacetate may then be
these glucogenic metabolites is oxaloacetate, converted to phosphoenolpyruvate and on to
which is converted to phosphoenolpyruvate glucose in the cytoplasm.
by phosphoenolpyruvate carboxykinase. The succinate produced in glyoxysomes can
As has been seen, the main fate of mito- also be used to produce oxaloacetate. However,
chondrial acetyl-CoA is to undergo condensa- the glyoxysomes do not contain the TCA cycle
tion with oxaloacetate to form citrate. The net enzymes required to bring about this conver-
synthesis of glucose from acetyl-CoA is impos- sion, and the succinate must be transferred to
sible because two carbon atoms are lost as CO2 the mitochondria before it can be metabolized
in the reactions catalysed by isocitrate dehy- to oxaloacetate. In order to maintain the supply
drogenase and a-ketoglutarate dehydroge- of oxaloacetate for the glyoxylate cycle, mito-
nase. In order for glucose synthesis to occur chondrial oxaloacetate must be transferred back
from acetyl-CoA, the two reactions which pro- to the glyoxysomes. A barrier to this transfer is
duce CO2 must be by-passed. This is what the relative impermeability of the inner mito-
occurs in the glyoxylate cycle. chondrial membrane to oxaloacetate. In order
The reactions of the glyoxylate cycle are to overcome this problem oxaloacetate is
shown in Figure 11.13. The first two reactions transaminated to aspartate, which is then trans-
are identical to those of the TCA cycle, but ported to the glyoxysomes where it is convert-
isocitrate does not then undergo oxidative ed back to oxaloacetate (Figure 11.14). This
decarboxylation to a-ketoglutarate. Instead, it cycle is important during the germination of
is split by the enzyme isocitrate lyase into suc- many types of seeds where significant quanti-
cinate and the two-carbon compound, glyoxy- ties of lipid are stored. It enables these concen-
late. Glyoxylate then condenses with a mole- trated energy reserves to be converted to sugars
cule of acetyl-CoA to form malate which can which can be used to provide the building
be converted to oxaloacetate. Since no CO2 is blocks for the growing plant.
...
I~
Figure 11.13 The glyoxylate cycle.

The glyoxylate cycle 159
OxaJoacetate --+
... ,
/' ,
..
\
glucose malate
\
\ ( a-Ketoglutarate
"
\ I
\
\
\
Fumarate
.; ' "
Succinate
oxaloacetate
~alate
malate
, . ate
gtyo~
r/'"
SUCCIIl
\ I
isocitrate
citrate oxaloacetate
~
t J\-oxidation
~..coA
fatty' acids
fatty acids
Figure 11.14 The relationship between the metabolism of glyoxysomes and mitochondria. Isocitrate is
split into glyoxylate and succinate in the glyoxysomes. Glyoxylate is converted to malate, which is trans-
ported to the cytoplasm and used for glucose synthesis. Because glyoxysomes do not contain the
enzymes needed to regenerate oxaloacetate, succinate is transported to the mitochondria and converted
to oxaloacete which is transported back to the glyoxysomes as aspartate. In the glyoxysomes, aspartate is
converted back to oxaloacetate.
ELECTRON TRANSPORT AND OXIDATIVE 12
PHOSPHORYLATION

12.2 The mitochondrion 163
12.3 Components of the electron transport 163
chain
12.3.1 Flavoproteins 163
12.3.2 The iron-sulphur proteins 164
12.3.3 Ubiquinone 164
12.4 The electron transport chain complexes 164
12.4.1 Complex I - the NADH-dehydrogenase 164
complex
12.4.2 Complex II - the succinate 164
dehydrogenase complex
12.4.3 Complex III - the cytochrome b, c1 165
complex
12.4.4 Complex IV - cytochrome oxidase 165
12.5 Coupling of electron transport to ATP 166
synthesis
12.6 The yield of ATP 168
12.7 NADH produced in the cytoplasm enters 169
the electron transport chain via shuttle
reactions
12.8 Regulation of oxidative phosphorylation 169
by ADP/ATP supply
12.1 INTRODUCTION that once they are converted to their reduced

forms, intermediary metabolism will grind to a
In previous chapters we have seen that the halt unless there is a mechanism for their re-
catabolic processes such as glycolysis, the TCA oxidation. One such mechanism is the conver-
cycle and fatty acid oxidation bring about oxi- sion of pyruvate to lactate under anaerobic
dation of fuel molecules in order to release the conditions (Figure 12.1) (see also Chapter 16).
'trapped' chemical energy they contain. The However, under aerobic conditions the re-
oxidation reactions usually involve the trans- oxidation of NADH and FADH2 is brought
fer of electrons and/or hydrogen to acceptor about by a complex series of reactions located
molecules. A recurrent theme of oxidation in the inner mitochondrial membrane and
pathways is the reduction of NAD+ to NADH known as the electron transport chain. The
+ H+ and FAD to FADH2. The cell contains a overall effect of this process is the transfer of
finite amount of NAD+ and FAD. As they are electrons from NADH and FADH2 to oxygen,
essential for oxidative metabolism, it follows which is reduced to water:
162 Electron transport and oxidative phosphorylation
The nature of the link between electron
transport and ATP synthesis puzzled bio-
chemists for many years, and it was not until
the pioneering work of Peter Mitchell in the
Lactate 1960s on the movement of protons across the
Pyruvate dehydrogenase Lactate
inner mitochondrial membrane that a credible
Figure 12.1 The conversion of pyruvate to lactate mechanism linking electron transport and ATP
by lactate dehydrogenase. synthesis began to develop. In the intervening
years Mitchell's original ideas have been exten-
NADH + H+ + 7'202 ---> HzO + NAD+ sively tested, and it is now accepted that the
FADH2 + 7'202 ---> HzO + FAD electrochemical energy of a proton gradient
Electron transfer to oxygen is not direct but created across the inner mitochondrial mem-
takes place in an ordered sequence of discrete brane by the electron transport chain is used to
steps during which there is considerable release drive ATP synthesis. This mechanism is
of free energy. This energy is not wasted but is referred to as the chemiosmotic model of ATP
utilized by the mitochondrion to bring about synthesis. In fact, it is now known that
the phosphorylation of ADP to ATP. The over- chemiosmotic mechanisms are not only
all process of synthesis of ATP via the electron involved in ATP synthesis, but also in a num-
transport chain is referred to as oxidative phos- ber of energy-dependent processes such as
phorylation, and is summarized in Figure 12.2. active transport across membranes.
, ,~---------------------------------------------------, ,
I I
I
I
ACETYL-CoA
TCACYCLE
~T~ H20 ATP
I
I ~----~~~$------ FATlY ACIDS
.
\
...
' • • • - - - . . ACETYL-CoA
t
PYRWATE
t
GLUCOSE
Figure 12.2 The regeneration of NAD+ and FAD by the mitochondrial electron transport chain.
Mitochondria contain a limited supply of NAD+ and FAD. The reduced coenzymes produced by fatty
acid oxidation and the TeA cycle feed into the electron transport chain where they are oxidized. In the
process, ATP is synthesized. The oxidized coenzymes are re-used for oxidative metabolism.
Components of the electron transport chain 163
12.2 THE MITOCHONDRION them their brown colour. A number of different
cytochromes have been identified in the elec-
Mitochondria are surrounded by two mem-
tron transport chain. Cytochromes are globular
branes. The outer membrane has little bio-
proteins all of which contain iron porphyrin
chemical activity but acts as a partial barrier to
groups similar to those in haem. The different
the movement of molecules. It contains a pro-
tein, porin, which forms channels through the cytochromes, a, a3' bS6O' bS62' c and c1 are charac-
terized by the different subtypes of porphyrins
membrane allowing free movement of ions
they contain. The b cytochromes contain iron-
and molecules up to a molecular weight of
protoporphyrin IX, which is identical to that
approximately 10 000. The inner mitochondri-
found in haem and myoglobin. In cytochrome
al membrane, on the other hand, is a highly
types a and c the substituent groups attached to
selective permeability barrier and the move-
the porphyrin ring structure are slightly differ-
ment of ions and polar molecules across this
ent, the structures being referred to as haem A
membrane is tightly regulated by specific
and haem C, respectively.
translocases. The ratio of protein to lipid in this
The important feature of the cytochromes,
membrane (about 3:1) is higher than in any
which enables them to act as electron carriers
other cellular membrane and is a reflection of
is the presence of the iron atom which ca~
the biochemical activity of the membrane.
undergo oxidation and reduction between the
When observed under the electron micro-
Fe (II) and Fe (III) states (Figure 12.3).
scope it can be seen that the inner mitochon-
The a-type cytochromes also contain cop-
drial membrane is highly convoluted. The
per, which can participate in electron transfer
folds of the membrane are referred to as
reactions through transition between the Cu
cristae. It is probable that this extensive folding
(I) and Cu (II) oxidation states. These copper
evolved to increase the membrane surface
atoms play an important role in the final stage
area to allow for rapid exchange between the
of transfer of electrons to oxygen.
central compartment of the mitochondria, the
matrix, and the intermembrane space which
has a similar composition to the cytoplasm. 12.3.1 FLAVOPROTEINS
The processes of electron transport and oxida-
The dehydrogenase enzymes which initiate
tive phosphorylation are located in this mem-
the movement of electrons into the electron
brane. The mitochondrial matrix is the site of
transport chain are flavoproteins. NADH
the TCA cycle, fatty acid oxidation and amino
dehydrogenase, which catalyses the oxida-
acid metabolism.
tion of NADH, is a membrane-bound enzyme
12.3 COMPONENTS OF THE ELECTRON which contains flavin mononucleotide (FMN)
TRANSPORT CHAIN
as a prosthetic group. This enzyme accepts
electrons from NADH produced in the mito-
Extensive analysis of the inner mitochondrial chondrial matrix. Succinate dehydrogenase is
membrane has revealed much about the com- the only enzyme of the TCA cycle which is
ponents of the electron transport chain. In dir~ctly linked to the electron transport
broad terms, it is possible to group its compo- ~ham. It has FAD as its prosthetic group and
nents into four main categories. Each will be IS located on the inner surface of the inner
considered before describing the transfer of membrane.
protons from one component to another, and
how the various components are arranged
into a functional electron transport chain.
Fe (III) + e-·.::;;
..r===~b Fe (II)
Mitochondria are a rich source of Figure 12.3 The interconversion of iron between
cytochromes and it is these proteins which give oxidation states.
There are a number of other flavin-contain- 12.4 THE ELECTRON TRANSPORT CHAIN
ing enzymes which feed electrons into the COMPLEXES
electron transport chain. Acyl-CoA dehydro-
Studies of the inner mitochondrial membrane
genase, which catalyses the first reaction in the
have shown that electron transport is a highly
f3-oxidation of fatty acids, contains FAD and is
organised series of reactions which occur in a
located in the mitochondrial matrix. Glycerol-
fixed sequence. The various component parts
3-phosphate dehydrogenase is located on the
of the chain have been examined in detail, and
outer surface of the inner mitochondrial mem-
it has become clear that the process can be split
brane. This enzyme also contains FAD and
into four discrete stages which contain adja-
catalyses the oxidation of glycerol-3-phos-
cent groups of electron carriers and are
phate to dihydroxyacetone phosphate.
referred to as complexes I, II, III and IV.
12.3.2 THE IRON-SULPHUR PROTEINS

12.4.1 COMPLEX I - THE
These proteins differ from the cytochromes in NADH-DEHYDROGENASE COMPLEX
that, although they contain iron, they do not
Also known as NADH-ubiquinone oxidore-
contain haem. Instead, the iron atoms are
ductase, this complex is the largest of the four.
located between sulphur atoms of cysteine
It contains approximately 26 polypeptides and
residues of the protein and inorganic sulphide
at least seven iron-sulphur centres. It catalyses
to form a complex. The iron atoms function in
the transfer of electrons from NADH to the
the same way as in cytochromes, that is, they
FMN prosthetic group of NADH dehydroge-
act as electron carriers by undergoing cyclical
nase, their subsequent passage through the
oxidation and reduction. The iron and sulphur
iron-sulphur centres, and finally transfer to
occur as clusters or centres within the protein.
the mobile carrier ubiquinone. The orientation
In at least one case, that of succinate dehydro- of complex I in the inner mitochondrial mem-
genase, these iron-sulphur centres occur in
brane allows it to accept the hydrogens from
proteins which also contain other electron car-
NADH which are transferred to FMN, reduc-
riers, e.g. FAD, and may assist in the transfer of
ing it to FMNH2 • At this point the hydrogens
electrons from FAD.
and their associated electrons part company.
The protons are released into the intermem-
brane space while the electrons are passed via
12.3.3 UBIQUINONE
the iron-sulphur proteins to ubiquinone,
Ubiquinone, also referred to as coenzyme Q, where a further two protons are taken from
has a benzoquinone structure and is a lipid- the matrix to yield UQH2, reduced ubiquinone
soluble electron carrier. A number of different (Figure 12.4).
ubiquinones have been identified which differ In this way, complex I makes the first con-
in the length of their isoprenoid side chains. tribution to the development of the proton
The structure of ubiquinone is shown in gradient across the inner mitochondrial mem-
Figure 4.10. brane.
As will become clear below, ubiquinone
acts as an electron sink which links the trans-
12.4.2 COMPLEX 11- THE SUCCINATE
fer of electrons from NADH and from FADH2
DEHYDROGENASE COMPLEX
in succinate dehydrogenase, acyl-CoA dehy-
drogenase and glycerol-3-phosphate dehy- This complex, which is also known as succi-
drogenase to the rest of the electron transport nate-ubiquinone oxidoreductase, is linked
chain. directly to the TCA cycle via the enzyme succi-
The electron transport chain complexes 165
COMPlEX I
matrix
HAD+ NADH + H+
Figure 12.4 Complex I of the mitochondrial electron transport chain.
nate dehydrogenase. The complex contains complex in two one-electron steps, and the
two iron-sulphur proteins which accept elec- transfer of two protons into the intermem-
trons from the FADHz in succinate dehydroge- brane space. This complex makes an impor-
nase and pass them on to ubiquinone. The tant contribution to the transmembrane
transfer of electrons to ubiquinone is accom- potential. Finally, electrons pass from
panied by the uptake of two protons from the cytochrome c1 to cytochrome c, which is not
matrix. However, there is no evidence that this part of complex III, but is a peripheral protein
complex contributes to the transmembrane located on the outer surface of the inner
proton gradient, and while it is generally membrane and acts as the link between com-
accepted that complexes I, III and IV span the plexes III and IV (Figure 12.6).
inner membrane, it is likely that complex II
does not (Figure 12.5).
12.4.4 COMPLEX IV - CYTOCHROME OXIDASE
12.4.3 COMPLEX III - THE CYTOCHROME B, c1 This complex contains cytochromes a and a3• It
COMPLEX differs from the other cytochrome-containing
complex, complex III, in that its cytochromes
This complex is also known as ubiquinone contain not only Fe atoms but also eu atoms,
cytochrome c oxidoreductase. Its net effect which are essential in the final reduction of
is to transfer electrons from reduced oxygen to water. The stoichiometry of the
ubiquinone to cytochrome c. The complex reaction suggests that complex IV catalyses the
contains three cytochromes, bS60' bS62 and c1, an transfer of four electrons. This is important
iron-sulphur protein, and at least six other because it allows the complete reduction of
proteins which together span the membrane. oxygen to water without the generation of
The transfer of electrons from ubiquinone to reactive intermediate products such as hydro-
cytochrome c involves a rather circuitous gen peroxide, HzOz' which can cause consider-
series of reactions known as the Q cycle, able damage within the cell.
involving two molecules of ubiquinone, the
transfer of electrons through the cytochrome 0z + 4H+ + 4e- ..... 2HzO
COMPlEX If
ilt.nnernlnl .. ..,..
Figure 12.5 Complex II of the mitochondrial electron transport chain.
This complex contributes to the proton 12.5 COUPLING OF ELECTRON TRANSPORT

gradient in two ways: firstly by the consump- TO ATP SYNTHESIS
tion of matrix protons during the reduction of The movement of electrons along the elec-
oxygen, and secondly by the direct transfer of tron transport chain results in a large release
protons across the membrane (Figure 12.7). of free energy. The energy is not used direct-
The overall arrangement of electron trans- ly to synthesize ATP, but is conserved as the
port complexes is shown in Figure 12.8. transmembrane proton gradient which is
COMPLEXIU
iltenllembrane
space
matrix
H+ H+
Figure 12.6 Complex III of the mitochondrial electron transport chain.

Coupling of electron transport to ATP synthesis 167
COMPlEXrv
Intennembfw ..
SJ*8
Figure 12.7 Complex IV of the mitochondrial electron transport chain.
coupled to the synthesis of ATP. The move- the intermembrane space is positively
ment of protons across the inner mitochondr- charged compared with the matrix. These
ial membrane has two major effects. Firstly, it two factors combine to create an electrochem-
creates a chemical gradient of protons across ical gradient across the inner mitochondrial
the membrane which can be measured in membrane which is referred to as the proton
terms of a difference in pH between the mito- motive force (PMF).
chondrial matrix and the intermembrane The PMF is maintained because the inner
space. Thus, the matrix is alkaline with mitochondrial membrane is impermeable to
respect to the intermembrane space. protons and does not allow them to re-enter
Secondly, because protons are charged ions, the matrix down their concentration gradient.
intermembrane
space
- +--+ .
. !t. . .. ;, ••• UQ ... . .. . .~ . .
Figure 12.8 The organisation of the mitochondrial electron transport chain in the inner mitochondrial
membrane, showing how protons are 'pumped' into the intermembrane space.
Instead the energy of the PMF is channelled catalyses the hydrolysis of ATP to ADP and Pi.
through an enzyme complex in the inner When isolated Fa and Fj units are reconstruct-
mitochondrial membrane known as the FaFj- ed in membrane vesicles they combine to
ATPase, which catalyses the synthesis of ATP form a functional enzyme complex capable of
from ADP and inorganic phosphate. synthesizing ATP in the presence of a proton
Examination of the inner mitochondrial mem- gradient.
brane under the electron microscope reveals The precise mechanism by which the FaFj-
an array of projections on its inner surface. ATPase catalyses the synthesis of ATP is not
Each projection has the appearance of a spher- known. It has been suggested that once syn-
ical unit linked to the membrane via a stalk. thesized, ATP remains tightly bound to the
Initially it was thought that these structures enzyme active site, blocking any further syn-
might be artefacts produced by the fixation thesis of ATP. The proton gradient appears to
process required to visualize the mitochondri- provide the driving force to release ATP from
al membrane, but since it has been possible to the enzyme, allowing further synthesis to take
isolate them, studies to examine their proper- place.
ties have confirmed that they correspond to
the FaFj-ATPase (Figure 12.9).
12.6 THE YIELD OF ATP
The structure of the enzyme complex is not
fully understood but it is recognised that it has Estimates of the amount of ATP synthesized
two main components. The Fa unit (so named when electrons pass from NADH to oxygen
because it confers oligomycin sensitivity on vary slightly. It is generally agreed that around
the enzyme) acts as an anchor point within the 10 protons are transported across the inner
membrane for the Fj unit and forms a channel mitochondrial membrane for each molecule of
through which protons can return to the mito- NADH oxidized. It is estimated that three pro-
chondrial matrix. tons must pass through the FaFj-ATPase dur-
The Fj unit contains the catalytic site for ing the synthesis of each molecule of ATP. In
ATP synthesis. When isolated from the mem- addition, one proton is involved in the mem-
brane, the Fj unit cannot synthesize ATP but brane transport of ADP and Pi into the mito-
chondrial matrix, and of ATP into the cyto-
plasm. Thus the number of molecules of ATP
intermembrane apace synthesized per molecule of NADH oxidized
can be calculated at between 2.5 and 3.3
(depending on whether the number of pro-
tons used is taken as three or four). Similarly,
during the transfer of electrons from FADH2 to
oxygen, six protons are transported into the
intermembrane space, giving an estimated
yield of ATP per molecule of FADH2 oxidized
of 1.5-2.0. These figures agree well with the
laboratory measurements. For most purposes
it is normally assumed that the ATP yield for
NADH and FADH2 is three and two, respec-
matrix tively. This fits well with the view that there
F, Unit are three coupling sites in the electron trans-
port chain between complexes I and IV, and
only two coupling sites between complexes II
Figure 12.9 The structure of FIFo-ATPase. and IV.
Regulation of oxidative phosphorylation by ADP/ATP supply 169
12.7 NADH PRODUCED IN THE CYTOPLASM III of the electron transport chain (Figure
ENTERS THE ELECTRON TRANSPORT CHAIN 12.11).
VIA SHUTTLE REACTIONS NADH entering the mitochondria via the
malate-aspartate shuttle feeds into the elec-
Not all of the NADH oxidized by the electron tron transport chain via complex I and can
transport chain is produced in the mitochondr- therefore yield three molecules of ATP, where-
ial matrix; some is produced in the cytoplasm. as protons from NADH passing via the glyc-
However, the inner mitochondrial membrane erol-3-phosphate shuttle enter the electron
is impermeable to NADH. This problem is transport chain via complex III, resulting in
overcome by use of shuttle reactions. the production of only two molecules of ATP.
In the malate-aspartate shuttle, oxaloac-
etate is reduced to malate in the cytoplasm by
12.8 REGULATION OF OXIDATIVE
malate dehydrogenase. In this reaction, cyto-
PHOSPHORYLATION BY ADP/ATP SUPPLY
plasmic NADH is oxidized to NAD+. The
malate is transported into the mitochondrial Measurement of the P/O ratio of mitochondria
matrix via a membrane carrier which demonstrates the tight coupling between elec-
exchanges cytoplasmic malate for mitochondr- tron transport and ATP synthesis in freshly
ial a-ketoglutarate. Once in the mitochondria, prepared mitochondria. This ratio, measured
NADH is regenerated by conversion of malate using an oxygen electrode, indicates the quan-
back to oxaloacetate by mitochondrial malate tity of ATP synthesized per atom of oxygen
dehydrogenase. Thus the net effect of this part reduced to water. When mitochondria are
of the shuttle is the transfer of NADH from the given an oxidizable substrate such as malate or
cytoplasm to the mitochondrial matrix. The succinate and an excess of inorganic phos-
shuttle cycle is completed by a transamination phate, they will consume oxygen at a rate
reaction in which a-ketoglutarate is converted dependent on the supply of ADP.
to glutamate and oxaloacetate is converted to When ADP supply is very low the rate of
aspartate. This reaction is catalysed by aspar- oxygen consumption is also very low.
tate aminotransferase. The aspartate produced However, if ADP is added to the mitochondrial
is transported to the cytoplasm in exchange suspension there is a burst of oxygen consump-
for glutamate, and is used to regenerate cyto- tion which lasts until the ADP has been con-
plasmic oxaloacetate by a reversal of the sumed, i.e. converted to ATP. This regulation
transamination reaction catalysed by cytoplas- by ADP supply is called respiratory control, and
mic aspartate aminotransferase (Figure 12.10). it depends on the maintenance of the integrity
Cytoplasmic NADH can also feed into the of the inner mitochondrial membrane. As mito-
electron transport chain via the glycerol-3- chondrial preparations age, respiratory control
phosphate shuttle, in which cytoplasmic glyc- is gradually lost as the inner membrane
erol-3-phosphate dehydrogenase catalyses becomes 'leaky' and the proton gradient is dis-
the reduction of dihydroxyacetone phosphate sipated. Similarly, if mitochondrial membranes
to glycerol-3-phosphate. Glycerol-3-phos- are damaged by chemical or mechanical treat-
phate can pass freely from the cytoplasm to ment, respiratory control is lost.
the mitochondrial intermembrane space, Mitochondria that exhibit tight respiratory
where it undergoes oxidation to dihydroxy- control are said to be 'coupled', that is the
acetone phosphate by a mitochondrial glyc- process of electron transport is governed by
erol-3-phosphate dehydrogenase located on the rate at which ATP is synthesized, which in
the outer surface of the inner mitochondrial turn is proportional to the [ATP]/[ADP] ratio of
membrane. The enzyme contains FAD as its the cell. Many compounds have been identi-
prosthetic group and links directly to complex fied which in some way break the link
CYTOPLASM
~=.::; ~.
-----~
NADH+H+
Figure 12.10 The reactions of the malate-aspartate shuttle.
between electron transport and ATP synthesis. which specifically inhibit electron transport or
These compounds are referred to as 'uncou- ATP synthesis, in that their commOn mode of
pIers'. They are distinct from compounds action is to destroy the transmembrane proton
NAO+ NAOH + H+
[~.~~~~----J phoephaIe phoephate [

..... .~
ItfIocItoniIrII
~
••
COMPLEX·II
MITOCHONDRIAL
MATRIX
Figure 12.11 The reactions of the glycerol-3-phosphate shuttle and its relationship to complex III.
Regulation of oxidative phosphorylation by ADP/ATP supply 171
gradient (Figure 12.12). A classical example of which is important in the process of non-shiv-
a synthetic uncoupler is 2,4-dinitrophenol, a ering thermogenesis, particularly in neonatal
lipid-soluble weak acid which can penetrate animals. This type of adipose tissue is brown
the inner mitochondrial membrane, and acts due to the presence of unusually high num-
as a proton shuttle. The ionophore valino- bers of mitochondria. The function of this tis-
mycin can also act as an un coupler, not by sue is to oxidize fatty acids, not for ATP syn-
allowing protons to re-enter the matrix, but by thesis, but for heat production. This is
providing a mechanism for the influx of K+ achieved by the presence of thermogenin in
ions into the matrix. This influx of positively the inner mitochondrial membrane, which
charged ions reduces the electrical potential provides hydrophilic channels in the mem-
across the membrane and reduces the effec- brane through which protons can pass. The
tiveness of the PMF. energy released by electron transport in these
Thermogenin, a naturally occurring uncou- mitochondria is dissipated as heat via the
pIer, has received much attention. It is found extensive network of capillaries which perfuse
in the mitochondria of brown adipose tissue the tissue.
High [H+]
H+ H+
H+ --,
H+ H+
~
intermembrane
space
,.
~--------------~~~
,
Fof 1-ATPase
matrix
ADP I ATP
+Pi
+
H+
Figure 12.12 The overall process of oxidative phosphorylation showing how ATP synthesis occurs when
protons in the intermembrane space may flow back to the matrix via FaFl-ATPase, and how uncouplers
destroy the proton gradient.
FATTY ACID OXIDATION AND LIPID 13
BREAKDOWN

13.2 13-0xidation 174
13.2.1 Mitochondriall3-oxidation in animal 174
tissues
13.2.2 The reactions of l3-oxidation 176
13.2.3 13-0xidation of odd-numbered acids 176
13.2.4 13-0xidation of unsaturated acids 178
13.2.5 13-0xidation in peroxisomes and 179
glyoxisomes
13.2.6 The formation of ketone bodies 181
13.3 a-Oxidation 182
13.4 w-Oxidation 183
13.5 Peroxidation of fatty acids 183
13.5.1 Chemistry of lipid peroxidation 184
13.5.2 Prevention of fatty acid peroxidation 185
13.5.3 Detection and measurement of lipid 185
peroxidation
13.5.4 Effects of peroxidation in living 186
organisms
13.5.5 Lipoxygenase and cydo-oxygenase 188
13.6 Breakdown of lipids 189
13.6.1 Triacylglycerol breakdown 190
13.6.2 Phospholipid breakdown 191
13.6.3 Breakdown of glycolipids 192
13.1 INTRODUCTION oxidation in the TCA cyde leads to the pro-

duction of large quantities of the reduced
Fatty acids can undergo a number of oxidative coenzymes, NADH and FADH 2, which are
modifications. Quantitatively the most impor- used for the production of ATP. Additionally,
tant pathway of fatty acid oxidation is referred in plants, the l3-oxidation of fatty acids can be
to as the l3-oxidation pathway. This process used to provide acetyl-CoA for carbohydrate
occurs in all organisms and is the main route synthesis via the glyoxylate pathway (Chapter
by which fatty acids are utilized for energy 11). Two other oxidation processes, known as
production. In l3-oxidation, fatty acids are a-oxidation and w-oxidation, are involved in
degraded to smaller compounds, usually the specific modification of fatty acids, the for-
acetyl-CoA, which can then be oxidized via mer required to oxidize fatty acids at carbon 2,
the TCA cyde. The conversion of long-chain the latter oxidizing fatty acids at the methyl
fatty acids to acetyl-CoA and its subsequent terminal or w-carbon.
174 Fatty acid oxidation and lipid breakdown
Another type of fatty acid oxidation occurs transport around the body in the blood they
during the synthesis of the eicosanoids. This may be present as non-esterified fatty acids
group of fatty acid derivatives, considered to be (NEF As) bound to the carrier protein albumin,
'local hormones' in animals, are produced from or contained in phospholipids and triacyglyc-
the fatty acids all-cis a8,11,14 C20:3, all-cis a5,8,11,14 erols incorporated in lipoproteins, mainly chy-
C20:4 and all-cis a5,8,11,14,17 C20:5 by the action of lomicrons and very low-density lipoproteins
either a lipoxygenase or a cydo-oxygenase. (VLDL).
Unsaturated fatty acids also undergo non- Fatty acids in lipoprotein-bound lipids are
enzymic peroxidation. This process is usually released into the blood by the action of
free radial-mediated, and the susceptibility of lipoprotein lipase found on the luminal sur-
fatty acids to peroxidative breakdown is face of the capillary endothelial cells. NEF As
directly proportional to the number of double pass from the blood into adjacent cells both by
bonds present. Peroxidation can be both detri- diffusion and apparently by a membrane-
mental and beneficial. The 'off smells and mediated process.
flavours associated with fatty foods are due to In the cytoplasm, fatty acids must be acti-
the build-up of short-chain aldehydes and vated to their CoA ester derivatives before
ketones with unpleasant odours. However, they can be further metabolized. The reaction
some of the pleasant aromas and tastes associ- requires ATP and is catalysed by enzymes
ated with fresh fruit and vegetables are also called acyl-CoA synthetases (Figure 13.1).
attributed to volatile derivatives of fatty acids. There are a number of different enzymes all of
which catalyse the same general reaction but
which have differing chain-length specifici-
13.2 p.,OXIDATION
ties. The two most important in fatty acid oxi-
Most tissues can oxidize fatty acids by f3-oxidation are:
dation. There are two main sites of f3-oxidation
• medium-chain acyl-CoA synthetase (C4-
in the cell. In animals, the mitochondrial
C12);
matrix is the site where fatty acids are com-
• long-chain acyl-CoA synthetase (C10+).
pletely oxidized to acetyl-CoA. Peroxisomal
oxidation of fatty acids is important in the liver Long-chain acyl-CoA synthetase is a mem-
and kidney. f3-0xidation in peroxisomes brane-bound enzyme found on the peroxi-
appears to be a mechanism for shortening the some membrane, the endoplasmic reticulum
chain length of fatty acids, which may then be and the outer mitochondrial membrane. This
further oxidized in the mitochondrial matrix. distribution allows fatty acids to be metabo-
In plant leaf tissue, peroxisomes rather than lized by various pathways at different subcel-
mitochondria are the main sites of fatty acid lular sites, i.e. chain shortening in peroxisomes,
oxidation, whereas in seeds, glyoxysomes are phospholipid and triacylglycerol synthesis on
the major sites of fatty acid oxidation where the endoplasmic reticulum, and f3-oxidation in
the product of oxidation, acetyl-CoA, can feed the mitochondria.
directly into the glyoxylate pathway. The inner mitochondrial membrane pre-
sents a barrier to fatty acyl-CoAs destined for
oxidation. Neither CoA itself nor esters of CoA
13.2.1 MITOCHONDRIAL ,,-OXIDATION IN
can pass through this membrane. This is large-
ANIMAL TISSUES
ly due to the large size and charged nature of
Fatty acids metabolized by the f3-oxidation the CoA molecule (see Figure 19.6).
pathway may come from both exogenous To facilitate the movement of fatty acids
sources (i.e. dietary) or endogenous sources across the inner mitochondrial membrane to
(i.e. from adipose tissue and liver). During the mitochondrial matrix, acyl-CoAs are con-
~:~
RCOOH ReOOH
~~
HSCoA o
HSCoA+ATP •
RC-CARNITINE
AMP+PPI CARNITINE CARNITINE RCSCoA

~
l' l'
RCSCoA f,J .. RCSCoA
CELL OUTER INNER

MEMBRANE MITOCHONDRIAl. MITOCHONDRIAL
MEMBRANE AfE.ftIBRANE
Figure 13.1 Activation and transport of fatty acids to the mitchondrial matrix.
verted to acyl-carnitines by the enzyme carni- and their transfer to NAD+. The product of the
tine acyltransferase I located in the mitochon- reaction is 3-ketoacyl-CoA (Figure 13.2).
drial intermembrane space. Fatty acyl-carnitine The final step in the reaction cycle of l3-oxi-
derivatives cross the inner membrane in dation is the thiolytic cleavage of 3-ketoacyl-
exchange for carnitine by means of a transport CoA between carbons 2 and 3. The result is the
protein, acyl-carnitine translocase. In the mito- production of a molecule of acetyl-CoA and a
chondrial matrix fatty acyl-carnitines are con- fatty acyl-CoA two carbon atoms shorter than
verted back to CoA derivatives by carnitine the fatty acid which started the cycle. The reac-
acyltransferase II. Once in the mitochondrial tion is catalysed by the enzyme acetyl-CoA
matrix as CoA esters, fatty acids can undergo 13- acyltransferase, also known as l3-ketothiolase.
oxidation (Figure 13.1). This enzyme is active against substrates vary-
ing in chain length from 4 to 18 carbons. In
addition, a second enzyme, acetoacetyl-CoA
13.2.2 THE REACTIONS OF !3-0XIDATION
thiolase, is also found in mitochondria. It is
The l3-oxidation pathway uses a sequence of involved in the metabolism of the ketone
four reactions to remove a two-carbon unit body, acetoacetyl-CoA, in tissues such as heart
from the carboxyl end of the fatty acid. This muscle which use ketone bodies as an energy
reaction sequence is repeated until the fatty source, and in liver, a major site of ketone
acid is completely oxidized. This process is body synthesis (Figure 13.2).
summarized in Figure 13.2. The two major fuels used by cells to synthe-
The first step in the l3-oxidation of a fatty size ATP are glucose and fatty acids. Long-chain
acyl-CoA is the removal of a hydrogen atom fatty acids are highly reduced compounds and
from each of carbons 2 and 3 to yield a LV trans- therefore their complete oxidation to CO 2 and
enoyl-CoA. The hydrogen atoms are accepted water can lead to the production of large
by FAD to give FADH2 and the reaction is amounts of ATP. The complete l3-oxidation of 1
catalysed by a group of enzymes called acyl- mole of palmitic acid results in the production of
CoA dehydrogenases (Figure 13.2). In the 8 moles of acetyl-CoA which can be further oxi-
complete oxidation of palmitic acid, this reac- dized via the TCA cycle. The ATP yield from
tion takes place seven times. To cope with the palmitic acid oxidation is shown in Table 13.1.
decrease in the chain length of the fatty acyl- One molecule of ATP is hydrolysed to AMP
CoA as it is oxidized at least three acyl-CoA and PP i during the activation of a fatty acid
dehydrogenases are required. prior to transport into the mitochondrial
The next step in the oxidation process is the matrix. This is equivalent to the utilization of 2
addition of a molecule of water across the dou- ATP, thus there is a net synthesis of 129 moles
ble bond (Figure 13.2). The enzyme responsi- of ATP from the l3-oxidation of 1 mole of
ble for this reaction is enoyl-CoA hydratase. palmitic acid.
This enzyme, which has a broad chain-length
specificity, catalyses the stereospecific addi-
13.2.3 !3-0XIDATION OF ODD-NUMBERED
tion of water across the double bond. Thus,
ACIDS
the hydration of a trans double bond yields the
L-3-hydroxyacyl-CoA (Figure 13.2). The l3-oxidation of odd-numbered straight-
The third enzyme in the sequence, L-3- chain fatty acids is almost identical to those
hydroxyacyl-CoA dehydrogenase, is relatively containing an even number of carbon atoms.
non-specific with respect to chain length but is The process proceeds by the sequential
absolutely specific for the L-stereoisomer of the removal of two carbon units, acetyl-CoA, until
3-hydroxyacyl-CoA. The enzyme catalyses the a five-carbon intermediate remains. This is
removal of two hydrogen atoms from carbon 1 then converted into one molecule of acetyl-
CH3(CH2)12CH2CH2COSCoA
hexadecanoyl-CoA
CH 3COSCoA
FAD
acetyl-CoA HSCoA CH3(CH2)10CH2CH2COSCoA -----1
p-ketothio/ase tetradecanoyl-CoA acyl-GoA
dehydrogenase
CH3(CH2)aCH2CH2COSCoA - - - I
FADH2
;f dodecanoyl-CoA
/
-- "'4--,,- .----+ etc H
I
CH3(CH2)12nCH2COSCoA 1
CH 3(CH 2)12Y=C COSCoA
o ~-Oxidation ~
3-ketoacyl-CoA t!2..trans enoyl-CoA
(3-ketohexadecanoyl-CoA) 1 (",.2-trans hexadecenoyl-CoA)
~-----------------------y
1
~------------------------------y
NADH+H+
eooy/-GoA
L-3-hydroxyacyl-GoA hydratase
dehydrogenase
CH3(CH2)12yHCH2COSCOA
NAD+ OH
H20
L-3-hydroxyacyl-CoA
(L-3-hydroxyhexadecanoyl-CoA)
Figure 13.2 The overall scheme of I)-oxidation.

Table 13.1 ATP yield from the oxidation of palmitic acid
Pathway Net conversion Cofactors ATP yield

produced
[3-oxidation palmitic acid to 8 acetyl-CoA 7NADH - 21 ATP

7FADH2 - 14 ATP
TCA cycle 8 acetyl-CoA to CO 2 and water 24NADH - 72 ATP
8 FADH2 - 16ATP
8GTP - 8ATP
Total == 131 ATP
CoA and one molecule of propionyl-CoA. mal pathway of l3-oxidation. This results in
Since propionyl-CoA is a three-carbon com- the production of a 12-carbon enoyl-CoA
pound it can be utilized for glucose synthesis; intermediate (!l.3-cis dodecenoyl-CoA) in
however, the quantity of odd-numbered fatty which the double bond is in the wrong posi-
acids found in animal tissues is usually small tion, !l.3 instead of !l.2, and in the wrong con-
(1-2%) and therefore the contribution they figuration, cis instead of trans. The position
make to glucose production is negligible com- and configuration of the double bond are
pared with other glucogenic substrates. This is modified by the action of the enzyme enoyl-
one of the few occasions in animals when a CoA isomerase. This enzyme converts the !l.3
product of fatty acid oxidation can be convert- cis double bond into a !l.2 trans double bond
ed to glucose. which allows l3-oxidation to continue to com-
pletion (Figure 13.3).
The l3-oxidation of linoleic acid (!l.9,12C18:2)
13.2.4 [3-0XIDATION OF UNSATURATED ACIDS
requires yet another enzyme. The oxidation
A slightly modified version of the l3-oxidation process proceeds as for oleic acid (described
scheme is required to oxidize unsaturated above), however, following the action of
fatty acids. Figure 13.2 shows that the unsatu- enoyl-CoA isomerase only one further cycle
rated intermediate produced by acyl-CoA of l3-oxidation takes place before a la-carbon
dehydrogenase has the !l.2-trans configuration, intermediate with a cis double bond between
whereas most naturally occurring unsaturated carbons 4 and 5 (!l.4 decenoyl-CoA) is formed.
fatty acids contain double bonds with the cis This undergoes dehydrogenation in the first
configuration. As unsaturated fatty acids are step of the l3-oxidation cycle, catalysed by
degraded by removal of two carbon units from acyl-CoA dehydrogenase, to produce !l.2 trans,
the carboxyl end, the position of the double !l.4 cis-decadienoyl-CoA. This conjugated dou-
bonds moves relative to the carboxyl carbon. ble-bond system is then transformed by the
The carboxyl carbon is always carbon 1, so as enzyme 2,4-dienoyl-CoA reductase to yield
the fatty acid becomes shorter the position of !l.3 trans-decenoyl-CoA. Finally, the !l.3 trans
any double bond approaches carbon 1. This double bond is converted to a !l.2 trans double
has two important consequences in the l3-oxi- bond by enoyl-CoA isomerase, which allows
dation of unsaturated fatty acids. l3-oxidation to contihue to completion (Figure
For a fatty acid like oleic acid with one cis 13.4).
double bond between carbons 9 and 10, one By the use of these two additional enzymes,
additional reaction is required to complete 13- enoyl-CoA isomerase and 2,4-dienoyl-CoA
oxidation. The first stage in the process reductase, almost all naturally occurring
involves the removal of six carbon atoms unsaturated fatty acids can be oxidized via the
(three molecules of acetyl-CoA) by the nor- l3-oxidation pathway.
f3-oxidation 179
&9-cis-octadecenoic acid
(oleic acid)
3 cycles of p-oxidatlon l1~ 3 acetyl-CoA
&3-cis-dodecenoic acid
CoA
&2..trans-dodecenoic acid
5 cycles of ~-oxidation
6 acetyl-CoA
Figure 13.3 The (3-oxidation of oleoyl-CoA.
13.2.5 (3-0XIDATION IN PEROXISOMES AND enzymes in the different subcellular

GL YOXISOMES organelles. The membranes of peroxisomes
and glyoxisomes do not present a permeabili-
The l3-oxidation of fatty acids in peroxisomes ty barrier to the CoA derivatives of fatty acids
or glyoxisomes differs from that in mitochon- as does the inner mitochondrial membrane,
dria. The first reaction is catalysed by acyl-CoA thus there is no requirement for fatty acids to
oxidase in peroxisomes and glyoxisomes. This be converted to their carnitine derivatives
reaction produces hydrogen peroxide, which before they are oxidized. Unlike mitochondria,
is then broken down by the action of catalase. peroxisomes and glyoxisomes do not contain
The remaining reactions of the l3-oxidation an electron transport chain capable of utilizing
cycle appear to be the same as those in the the NADH produced by L-3-hydroxyacyl-CoA
mitochondria, although there may be structur- dehydrogenase. In order to ensure a supply of
al and functional differences between the NAD+ for continued fatty acid oxidation,
a9,12-cis-octadecadienoic acid
(linolenic acid)
3 oycles of ,..".,...,. ~ 3 acetyI-CoA

CoA
a3.8-cis-dodecadienoic acid
1~~oAt
-..-:" CoA
t,2..trans, a8-cis-dodecadienoic acid
1 cycle of _ ~ 1 aceIyI-CoA
C~COA
t,2-trans, t,.-cis-decadienoic acid
~
NADPH+H+
2,4-dienoyl-CoA reductase
NADP+
CH 3
~CoA
t,3-trans-decenoic acid
~~~~
1-~'-
CH 3 + + + +
~oA
A2-trans-decenoic acid
4 oycIes of - 1
5 acetyl-eoA
Figure 13.4 The l3-oxidation of linoleoyl-CoA.

{3-oxidation 181
NADH is transported to the cytoplasm in animals are said to be suffering from ketosis.
exchange for NAD+. The synthesis of ketone bodies, referred to as
In animals, peroxisomal oxidation makes a ketogenesis, occurs mainly in the liver.
significant contribution to overall fatty acid oxi- Ketogenesis occurs when the supply of
dation: in liver this may be as high as 50% of the oxaloacetate is insufficient to allow all of the
total fatty acid oxidation. However, only partial acetyl-CoA produced in the mitochondria to
oxidation of fatty acids occurs in peroxisomes. enter the TCA cycle. It is a mechanism which
These organelles appear to be particularly allows acetyl-CoA to be converted to useful
important in conversion of long-chain fatty compounds which can be exported from the
acids to medium-chain-length products which liver and used by other tissues as an energy
are then converted to acyl-carnitine derivatives source. However, in certain circumstances
and transported to the mitochondrial matrix, which can arise in pregnant sheep and lactat-
where the oxidation process is completed. The ing cows, the concentration of ketone bodies
acetyl-CoA produced in the peroxisome is also in the blood becomes too high and can lead to
transported to the mitochondrial matrix where the development of clinical conditions known
it can be oxidized in the TCA cycle or converted as pregnancy toxaemia and bovine ketosis.
to ketone bodies. The movement of acetate The pathway of ketone body synthesis
between these subcellular organelles is via a occurs mainly in the mitochondrial matrix. The
carnitine-mediated mechanism. initial steps of ketone body production are
In plants, glyoxisomes in the seeds and per- identical to those of the synthesis of cholesterol
oxisomes in the leaf are the primary sites of described in Chapter 19. Two molecules of
~-oxidation. Indeed, it is now thought that acetyl-CoA condense to form acetoacetyl-CoA
mitochondrial ~-oxidation makes only a very by a reversal of the ~-ketothiolase reaction of ~
small contribution to the oxidation of fatty acids oxidation. The acetoacetyl-CoA then reacts
in plants, and it is clear that both glyoxisomes with another molecule of acetyl-CoA to form
and peroxisomes are capable of complete oxi- hydroxymethylglutaryl-CoA (HMG-CoA). This
dation of fatty acids. In some germinating seeds HMG-CoA is hydrolysed to yield one molecule
the quantity of fatty acids mobilized from of acetoacetate and one molecule of acetyl-CoA.
stored fat and oxidized via the glyoxisomes is Some acetoacetate is reduced to ~-hydroxybu
large, particularly in oil-seed varieties. These tyrate by ~-hydroxybutyrate dehydrogenase.
specialized subcellular organelles contain both The extent of this reduction is determined
the enzymes required to oxidize fatty acids to largely by the ratio of NAD+ to NADH in the
acetyl-CoA, and the enzymes of the glyoxylate liver. Both acetoacetate and ~-hydroxybutyrate
pathway, isocitrate lyase and malate synthase, can leave the mitochondria and enter the
which enable plants to convert the acetyl-CoA
OH 0
to oxaloacetate and on to glucose. I II
CH;CH-CH~-oH ~-Hydroxybutyrate
13.2.6 THE FORMATION OF KETONE BODIES
There are three principal ketone bodies, ~

Acetoacetate
hydroxybutyrate, acetoacetate and acetone
(Figure 13.5). These compounds are normally
present in blood in trace amounts, except in
ruminants where ~-hydroxybutyrate is a prod-
Acetone
uct of butyrate metabolism as it passes across
the rumen wall. When their plasma concentra- Figure 13.5 The structure of the ketone bodies: (a)
tions increase significantly above the norm, l3-hydroxybutyrate; (b) acetoacetate; (c) acetone.
bloodstream. Acetoacetate may undergo spon- and/or breakdown of the a-hydroxy fatty
taneous decomposition to produce acetone, acids found in certain types of tissue lipids,
which can often be smelled on the breath of particularly in brain tissue where the sphin-
ketotic animals (Figure 13.6). golipid fraction contains a large amount of
this type of fatty acid. Thirdly, it may act in
concert with [3-oxidation, to facilitate the oxi-
13.3 a-OXIDATION
dation of fatty acids with structural features
As its name suggests, this process is the oxida- which prevent oxidation by [3-oxidation
tion of fatty acids at the a-carbon (carbon 2). It alone. An example of this combined action of
results in either the removal of a single carbon a- and [3-oxidation of fatty acids is the degra-
atom from the carboxyl end of a fatty acid or dation of phytanic acid (3,7,1l,15-tetramethyl
the production of a-hydroxy fatty acids palmitic acid) derived from phytol. The pres-
(Figure 13.7). ence of the methyl substituent on carbon 3
The metabolic significance of a-oxidation inhibits [3-oxidation; however, if the fatty acid
in animal tissues is still not understood. There is shortened at the carboxyl end by one car-
are three areas in which it may play an role. bon, [3-oxidation can proceed. Due to the posi-
Firstly, it may be the mechanism whereby tion of the methyl groups, the products of phy-
odd-numbered fatty acids are synthesized, i.e. tanic acid oxidation are alternate molecules of
by the removal of a carbon atom from the car- propionyl-CoA and acetyl-CoA. It is not clear
boxyl end of an even-numbered fatty acid. whether fatty acids undergo a-oxidation in the
Secondly, it may be involved in the synthesis free form or as CoA derivatives, or what is the
Hydroxymethylglutaryl-CoA
0 o 0 synthase OH 0
II p-ketothiolase
II II I II
2 CHj""C-SCoA
'\
~ CHj""C-CH~-SCoA ---7--:::OO--+~ HOOC-CH29-CH~-OH
( CH 3
HSCoA
Acetyl-CoA Acetoacetyl-CoA o Hydroxymethylglutaryl-CoA
II
CHj""C-SCoA
Acetyl-CoA
lyase
p-hydroxybutyrate 0 0
II 7" '\
dehydrogenase ~ II
~Hj""C-CH~-OH
II
p-Hydroxybutyrate Acetoacetate
NADH + H+ o
II
CHj""C-SCoA
1
Acetyl-CoA
Acetone
Figure 13.6 Pathway for the synthesis of ketone bodies.
Peroxidation of fatty acids 183
(a) shortening of a fatty acid by 1 carbon atom
CO2 + H20 NAO+ NADH
~ -\.~~4....
OOH
O2 I
- -•• RCHCOOH RCHO RCOOH
(b) production of an a-hydroxy fatty acid
OOH OH
I I
RCHCOOH RCHCOOH
Figure 13.7 a-oxidation of fatty acids.
exact subcellular location of the process. involving a specialized cytochrome (P450)' In

Nevertheless, it seems likely that the process is plants, the involvement of the cytochrome is
associated with the microsomal fraction. in doubt. w-Oxidation may be an essential
The importance of ex-oxidation in plants is step in the oxidation of fatty acids where the
also unclear but it has been demonstrated carboxyl end is unavailable for l3-oxidation, as
that, with the exception of the germinating the production of a carboxyl group from the
seed where l3-oxidation is very active, ex-oxi- methyl carbon of a fatty acid may allow the 13-
dation may be the most important pathway of oxidation process to start from the opposite
fatty acid oxidation. In addition to its role in end of the molecule. The production of w-
degradation of fatty acids, ex-oxidation is hydroxy fatty acids may have a role in the for-
almost certainly involved in the production of mation of cutin and suberin.
the long-chain fatty alcohols and hydrocar-
bons found in the cutin and suberin compo-
13.5 PEROXIDATION OF FATTY ACIDS
nents of the cuticle.
Food that is not stored under the correct condi-
tions will rapidly develop unpleasant
13.4 (!)-OXIDATION
organoleptic properties characterized by 'off'
In this process, fatty acids undergo oxidation flavours and smells. This is an extremely com-
at the w- or methyl carbon to form dicar- plex process involving the interaction of many
boxylic acids and w-hydroxy fatty acids. In physical, chemical and biological factors, the
animals, the enzyme responsible appears to combined effects of which are perceived pri-
be a mixed-function oxidase associated with marily as changes in the taste, smell and texture
the endoplasmic reticulum and probably of food.
The deterioration in food quality is often • propagation phase
linked t() enzymic and non-enzymic modifica- • termination phase.
tions of lipids and, in particular, to the fatty
acids they contain. Unsaturated fatty acids, In the initiation phase molecular oxygen, or
particularly PUFAs, are susceptible to non- a free radical such as the highly reactive
enzymic peroxidation. This process con- hydroxyl radical OH·, reacts with an unsatu-
tributes to the development of unpleasant rated fatty acid to form a fatty acyl free radical
odours and flavours associated with fatty by abstraction of hydrogen from a methylene
foods (rancidity). group adjacent to the double bond in the fatty
acid. The presence of pro-oxidant factors such
as transition metals (e.g. iron and copper),
13.5.1 CHEMISTRY OF LIPID PEROXIDATION ultraviolet or ionizing radiation promote this
initial stage of peroxidation:
Peroxidation is directly proportional to sub-
strate concentration and the partial pressure of RH + O 2 ---> R· + H0 2 •
oxygen. The rate of peroxidation also increases RH + OH· ---> R· + HzO
with the extent of existing fatty acid oxidation, Propagation occurs when the fatty acyl rad-
indicating that the process is autocatalytic; ical reacts with molecular oxygen to form a
hence it is often referred to as autoxidation. fatty acyl peroxide radical, which in turn initi-
Once started, a chain reaction is set up which ates new radical formation and results in the
perpetuates and accelerates the peroxidation production of a fatty acyl hydroperoxide.
process. The susceptibility of unsaturated fatty
acids to peroxidation is proportional to the R· + O 2 --> ROO·
number of double bonds they contain; thus ROO· + RH --> ROOH + R·
monounsaturated fatty acids undergo peroxi- Fatty acyl hydro peroxides decompose
dation at a slower rate than PUFAs. The rela- either to produce fatty acyl peroxide radicals
tionship between the degree of unsaturation of which further initiate peroxidation, or may be
a fatty acid and its susceptibility to peroxida- converted to alkoxy radicals. These transfor-
tion can be seen most clearly with pure fatty mations are greatly enhanced by the presence
acids. For fatty acids with 1,2,3,4,5 and 6 dou- of transition metals.
ble bonds, the relative rates of oxidation are
approximately 1, 40, 80, 160, 240 and 320, ROOH + M(1l+1)+ --> ROO· + Mn+ + H+
respectively. In fats or oils with a mixed fatty ROOH + Mn+ ---> RO· + OH- + M(n+l)+
acid composition, indicators of the degree of
unsaturation, such as the iodine value, can be Alkoxy radicals are unstable compounds
useful predictors of its likely susceptibility to which undergo chain fragmentation, termed
peroxidation. [3-scission, to produce alkyl radicals and low
The production of a fatty acid hydroperox- molecular-weight volatile aldehydes such as
ide proceeds via the formation of a fatty acyl octanal, non anal, 2-decenal and 2-undecenal
free radical, which arises due to the reaction of (Figure 13.8). Some of these compounds are
an unsaturated fatty acid with other free radical responsible for the unpleasant smells and
species. Fatty acyl free radicals can themselves tastes characteristic of rancid fatty foods.
interact with other unsaturated fatty acids to Termination occurs when radicals react
promote further free radical formation. together or with chain-breaking compounds
Three phases have been identified in the to produce stable molecules which do not ini-
peroxidation of fatty acids: tiate or propagate further peroxidation. Most
natural and synthetic antioxidants are chain-
• initiation phase breaking compounds and are themselves oxi-
0" RCHO+R'" clear that a-tocopherol plays an important role

I in the protection of membrane unsaturated
R-CH-R' fatty acids.
R"+R'CHO
The lipophilic phytyl side chain of a-toco-
Figure 13.8 j3-Scission of an alkoxy radical to pro- pherol is located in the hydrophobic lipid
duce an alkyl radical and an aldehyde. bilayer region of membranes (Figure 13.9). The
role of a-tocopherol appears to be two-fold:
dized and destroyed in the process. The firstly, to neutralize free radical species in the
length of the induction phase of oxidation can region of the membrane and thereby prevent
be extended by the addition of antioxidants, initiation of fatty acid peroxidation; and sec-
propagation being delayed until the antioxi- ondly, when fatty acid peroxide radicals have
dant activity is reduced. been formed, to break the chain of autoxida-
tion by converting the peroxide radical to a
hydroperoxide. It is estimated that about 90%
13.5.2 PREVENTION OF FATTY ACID
of the fatty acid peroxide radicals formed in
PEROXIDATION
membranes are neutralized by a-tocopherol
In biological systems, natural antioxidants are before they react with other PUFAs. Both of
present which minimize the extent to which these functions require that the a-tocopherol
fatty acid peroxidation takes place. The most has a higher affinity for free radicals than do
important of these are the group of com- unsaturated fatty acids.
pounds known collectively as vitamin E. In A number of synthetic antioxidants are
animals, the most commonly occurring of used by the animal feed and human food
these compounds is a-tocopherol. In plants, industries to minimize oxidative deterioration
and in particular in seed oils, the other isomers of lipids during processing and storage. The
of tocopherol and tocotrienol are present in structures of the most commonly used syn-
significant amounts; however the vitamin E thetic antioxidants are shown in Figure 13.10.
activity of these isomers is usually expressed
as a-tocopherol equivalents (Table 13.2).
13.5.3 DETECTION AND MEASUREMENT OF
The nature of the complex interactions of a-
LIPID PEROXIDATION
tocopherol with unsaturated fatty acids and
with other intracellular antioxidants such as Numerous methods have been developed to
ascorbic acid are slowly being unravelled. It is detect and quantify lipid peroxidation. Some,
Table 13.2 Tocopherol and tocotrienol content of common fats and oils
Tocopherols (mg) Tocotrienols (mg)

Fat/oil {3 y B a {3 Vitamin E activity as
a-tocopherol (mg)
Corn 112 50 602 19 198

Cottonseed 390 387 428
Palm 256 316 70 146 3 335
Soyabean 75 15 800 266 2 171
Wheat germ 1330 710 260 271 26 18 1736
Cod liver 220 220
Tallow 27 27
Adapted from Gunstone, F.D., Harwood, J.L. and Padley, F.B. (1994) The Lipid Handbook, 2nd edn,
Chapman & Hall, London.
Upid bilayer
Polar head groups

of phospholipids
1
Fatty acid
chains
Figure 13.9 Orientation of a-tocopherol in the membrane lipid bilayer.
such as measurement of changes in UV retarded. Diets containing fats with a PV in

absorbance due to migration of double bonds excess of 1200 induced rapid death. In poultry,
and formation of conjugated dienes during a number of reported cases of toxicity have
peroxidation, or estimation of the peroxide involved the feeding to birds of oils which
value (PV), measure peroxide formation. have been heated at high temperatures for
Others, such as the thiobarbituric acid test or prolonged periods. This leads not only to the
determination of the anisidine value, rely on formation of peroxides, but also to the produc-
measurement of the products of hydroperox- tion of polymerized fatty acid derivatives
ide breakdown. which are toxic. The use of recycled cooking
oil has been linked with increased mortality,
and with the incidence of burnt hocks as a
13.5.4 EFFECTS OF PEROXIDATION IN LIVING
result of the production of sticky excreta
ORGANISMS
caused by poor digestion of oxidized and
There is growing interest in the effects of lipid polymerized oil.
peroxidation in living organisms. It has been Membrane phospholipids and glycolipids
known for some time that peroxidized, highly are rich in PUFAs which are susceptible to
unsaturated oils are toxic in a number of ani- oxidative damage. Aerobic metabolism with-
mal species, and that the toxicity is propor- in cells generates active oxygen species, such
tional to peroxide content. For example, rats as hydrogen peroxide, hydroxyl radicals and
fed diets containing fat with a peroxide value superoxide radicals, which can react with
(PV) of 100 showed no adverse effects, where- membrane lipids producing peroxides and
as the growth of those fed diets containing fat peroxide-breakdown products. These oxida-
with PVs between 400 and 800 was severely tive changes can cause alterations in mem-
OH
¢f(CH~
OCH 3
Butylated hydroxyanisole (BHA) Butylated hydroxytoluene (BHT)
OH
¢f(CH~
OH
Tertiary butylated
Ethoxyquin
hydroxyquinone (TBHQ)
R = -(CH2)2CH3 propyl
R = -(CH2)3CH3 butyl
R = -(CH2)7CH3 octyl
R = -(CH2)11CH3 dodecyl
The Gallates
Figure 13.10 Commonly used synthetic antioxidants: butylated hydroxyanisole (BHA); butylated hydroxy-
toluene (BHT); tertiary butylated hydroxyquinone (TBHQ); ethoxyquin; the gallates.
brane structure and integrity which may system, the function of and communication
affect cellular function. For example, the reac- between the different cell types involved in
tion of fatty acid peroxides with membrane the coordinated immune response is mediat-
proteins may result in changes in receptor ed via the plasma membrane. Infection and
and transport protein-binding characteristics. disease often stimulate the production of free
The release and conversion of PUFAs to radicals which may lead to an increase in
prostaglandins and other eicosanoids may membrane lipid peroxidation and impair-
also be adversely affected. In the immune ment of the immune response. A number of
defence mechanisms are present within the In mammals, lipoxygenase and cydo-oxy-
cell to combat the adverse effects of these genase catalyse the initial steps in the conver-
reactive species, for example antioxidants sion of all-cis a 8,IU4 C20:3, all-cis a5,8,1l,14 C20:4
such as vitamin E, and metalloenzyme sys- and all-cis a5,8,IU4,17 C20:5 to leukotrienes,
tems such as glutathione peroxidase, super- prostaglandins, prostacydin and thrombox-
oxide dismutase and catalase. The immune anes, compounds collectively known as
response in diseased animals may be eicosanoids. Their structures are shown in
enhanced by nutritional supplementation Figure 13.11. Leukotrienes are produced by
with a-tocopherol and essential metallo- the action of lipoxygenase, whereas cydo-oxy-
enzyme minerals such as selenium, zinc genase modification of PUFAs leads to the
and manganese. synthesis of prostaglandins, prostacydin and
thromboxanes. The latter enzyme is a compo-
nent of the multifunctional protein, prosta-
13.5.5 LIPOXYGENASE AND CYCLO-
glandin endoperoxide synthetase (PES), PES
OXYGENASE
inhibitors indude several non-steroidal anti-
A number of lipoxygenase enzymes, contain- inflammatory drugs, e.g. aspirin and indo-
ing non-haem iron, occur in plant and animal methacin, which compete with the fatty acid
tissues. These enzymes catalyse the oxidation substrates for the enzyme active site. In the
of fatty acids by molecular oxygen. The imme- case of aspirin (acetylsalicylic acid) the active
diate product of the reaction is a hydroperox- site is irreversibly acetylated resulting in
ide which may be further metabolized to a enzyme inactivation.
variety of hydroxy derivatives and low-molec- The biochemical and physiological func-
ular-weight volatile compounds. tions of these fatty acid derivatives are the
In some plants, lipoxygenases are present in subject of considerable ongoing research.
large amounts, for example in yellow bean They appear to function as locally produced
and soyabean seeds (family Leguminosae) and hormones or signalling molecules, which
potato tubers (family Solanaceae). The soya- may have some effects at their site of pro-
bean enzyme is probably the best character- duction, but more generally are released into
ized. Lipoxygenase activity is of two types: the extracellular fluid and have effects in the
type I is active against free fatty acids, and local tissue environment They have very
type II which is most active against ester- short half-lives, in the order of seconds to
bound fatty acids. These enzymes are impor- minutes, and can have profound effects
tant in the development of tastes and flavours upon cellular activity at concentrations rang-
in plant tissues. These occur due to the further ing from 10-6 to 10-15 M, Most body tissues
metabolism of the initial hydroperoxide prod- and fluids contain minute quantities of
uct of lipoxygenase to low-molecular-weight eicosanoids, although human and sheep
aldehydes and organic acids such as hexanal, seminal fluid are particularly rich sources of
2-nonenal and 9-oxo-nonanoic acid, produced prostaglandins, the most common being
from linoleic acid. It has been suggested that PGE 2 and PGF2n •
short-chain products and other derivatives of The main physiological effects elicited by
hydroperoxides may act as growth regulators, eicosanoids are summarized in Table 13.3.
for example, in fruit ripening and seed germi- Because of the potential medical and veteri-
nation. Post-harvest, uncontrolled lipoxyge- nary uses of eicosanoids, there is considerable
nase activity can lead to the development of interest in the pharmaceutical industry in the
'off flavours, and heat treatment is often nec- development of longer-lived analogues.
essary to inactivate the enzymes. Probably the best known use of prostaglandin
~OOH~
Arachidonic acid ~
o
~COOH ~COOH
l;..~~
Leukolriene ~
OH
(LTA,v
OH
OH 1
~COOH ~COOH
HO~ HOAo~
Leukolriene B. OH
(LTB,v
HO OH
HO Proslac)din
o
~OH
HO OH ~
HO OH
Prostaglandin F2a
Prostaglandin ~
Figure 13.11 Leukotrienes and eicosanoids derived from arachidonic acid.
analogues in agriculture is in the synchroniza- 13.6 BREAKDOWN OF LIPIDS

tion of oestrus in farm animals. For example,
the synthetic analogues of PGF 2u such as clo- Both storage and structural lipids are in a con-
prostenol, given in two successive injections at stant state of turnover - they are continually
intervals of 10-12 days, result in the onset of being synthesized and broken down. This is a
oestrus about 3 days after the second injection. complex, regulated process which, within
Table 13.3 Summary of the main effects of acids is achieved by the migration of fatty acid
eicosanoids from the 2 position to the 1 position and its
subsequent release. The fatty acids released
Eicosanoid Effects are taken up by the glyoxysomes where they
PGEs Contraction of intestinal lon- are oxidized. The mechanism of transfer of
gitudinal muscle; relaxation fatty acids between the sites of storage and of
of sphincters; dilation of the oxidation is poorly understood, but in some
cervix seeds this may be achieved by direct contact
PGF2a Degeneration of the corpus between the oil droplet and the glyoxysome.
luteum; contraction of uterine
smooth muscle during partu- Animals
rition
Prostacyclin (PGI2) Vasodilator; inhibition of In adipose tissue, fatty acids are released from
platelet aggregation triacylglycerols by lipolysis. The release of
Thromboxane ~ Vasoconstrictor; stimulation fatty acids and their transport for use else-
of platelet aggregation where in the body is often referred to as fatty
Leukotrienes Chemotaxis, inflammatory
responses, bronchoconstric- acid mobilization. This requires the complete
tion, vasoconstriction, hydrolysis of triacylglycerols to fatty acids and
vasodilation glycerol. The fatty acids are transferred from
the adipocyte to the bloodstream where they
become bound to albumin and are then
highly organized organisms such as plants
referred to as non-esterified fatty acids
and animals, allows the type and quantity of
(NEFAs). As fatty acids released from adipose
individual lipids to be modified in response to
tissue triacylglycerols may be re-esterified
changes in nutritional and physiological cir-
without being released into the circulation, the
cumstances. The biosynthesis of complex
rate at which NEFAs enter the blood depends
lipids is discussed in Chapter 19. In the follow-
not only on the rate of lipolysis, but also on the
ing section the breakdown of triacylglycerols,
rate of esterification of fatty acids into triacyl-
phospholipids and glycolipids are described.
glycerols. Both lipolysis and esterification are
subject to acute and long-term regulation in
13.6.1 TRIACYLGLYCEROL BREAKDOWN response to changes in nutritional and physio-
logical status. In very simple terms, fatty acids
Plants
are deposited as triacylglycerols in adipose tis-
In some plants, large quantities of triacylglyc- sue when the rate of esterification exceeds that
erol are stored in seeds or fruits. In dry seeds of lipolysis, whereas fatty acids are released
prior to germination there is little enzymic into the blood when the rate of lipolysis
activity, however during imbibition (the exceeds that of esterification.
uptake of water by seeds during germination) The hydrolysis of a triacylglycerol occurs in
there is an increase in the activity of a number three stages. The rate-limiting step is the initial
of enzymes including triacylglycerol lipases. cleavage of triacylglycerol to diacylglycerol
Their substrates are contained in oil droplets and a fatty acid. This step is catalysed by the
(oleosomes) within the seed and the enzymes enzyme triacylglycerol lipase, sometimes also
act at the surface of the droplet, probably with called hormone-sensitive lipase, which is the
the help of binding proteins to facilitate the main site of regulation of lipolysis (see further
attachment process. The lipases catalyse the detail in Chapter 30). Diacylglycerols pro-
release of fatty acids esterified to the 1 and 3 duced in this reaction are further metabolized
position of triacylglycerols, to yield a mono- by other lipases to give fatty acids and glycerol
acylglycerol. Complete release of all three fatty (Figure 13.12).
Breakdown of lipids 191
Triacylglycerol (TriacylglyceroilipaSe )
l~D~+h*y_
L Monoacylglycerol + fatty acid
Figure 13.12 The sequential breakdown of triacylglycerols.

L GIyceroI+h*y_
13.6.2 PHOSPHOLIPID BREAKDOWN involved in the modification of the fatty acid

composition of existing membrane phospho-
Enzymes which are involved in the break- lipids. In animals, phospholipase Az is particu-
down and remodelling of phospholipids are larly important in the release of arachidonic
called phospholipases. In plant and animal tis- acid required for eicosanoid synthesis. Both
sues, a number of phospholipases have been enzymes are found in the secretions of the
identified which differ in the position at which digestive tract and are required for the diges-
they act on the phospholipid molecule. These tion of dietary phospholipids.
enzymes and their sites of action are shown in Phospholipase B appears to be present only
Figure 13.13. in microorganisms, and because it acts at both
Phospholipases Al and Az are widely dis- the 1 and 2 positions, it is able to act on both
tributed and are responsible for the removal of intact phospholipids and lysophospholipids.
fatty acids from intact phospholipids. The Phospholipase C has an important role in
resulting compound which contains only one the control of enzyme activity. Many enzymes
fatty acid, in either the 1 or 2 position, is called are regulated by a calcium-dependent phos-
a lysophospholipid. These enzymes are phorylation/dephosphorylation mechanism.
Phospholipase B
o
II
o CH~~
il
711~ CH;O=-P-:-O-X
Ph _ _ A,
--~ 7!~
Phospholipase C Phospholipase 0
Figure 13.13 The sites of action of phospholipases on a typical phospholipid.

The regulation process is initiated by .an 13.6.3 BREAKDOWN OF GL YCOLIPIDS
increase in the activity phospholipase C whIch
Plants are rich in glycolipids. Their catabolism
catalyses the hydrolysis of plasma membrane
is particularly active during senescence and
phosphatidylinositol-4,5-bisphosphate to pro-
following damage to tissues. Complete break-
duce inositol trisphosphate and diacylglycerol.
down occurs in two stages: firstly, the fatty
The former causes release of sequestered calci-
acids are removed by acyl hydrolase, and sec-
um. In addition to this regulatory function,
ondly, the sugar residues are removed by the
phospholipase C is also involved in the gener-
action of galactosidases. In grazing ruminants
al remodelling of tissue phospholipids. a large proportion of the ingested lipid is in
Although phospholipase D is found in ani- the form of glycolipids. These lipids are rapid-
mal tissues, the main source of this enzyme is ly broken down by the action of bacterial acyl
plant tissue. It is a very active enz'yme hydrolases and glycosidases.
involved in phosphatidate exchange reactIons;
however, it is not clear what its role is in phos-
pholipid metabolism.
BREAKDOWN OF PROTEINS AND THE 14
OXIDATION OF AMINO ACIDS

14.2 Breakdown of proteins 194
14.2.1 Digestion of proteins 194
14.2.2 Protein turnover 194
14.3 Breakdown of amino acids 194
14.3.1 Transamination reactions 194
14.3.2 Deamination 195
14.3.3 Oxidation of carbon skeletons of amino 195
acids
14.3.4 The fate of ammonia 197
14.3.5 The urea cycle 197
14.3.6 The fate of urea in ruminants 199
14.4 Precursor functions of amino acids 199
14.1 INTRODUCTION animals are frequently supplied with more

amino acids than are required for protein syn-
In living cells, proteins are generally unstable thesis and they have little capacity to store
compounds, and are continually broken down them, the excess must be degraded. Animals
and resynthesized. Proteins are easily dena- do this by oxidizing amino acids, obtaining
tured and even the most robust of them lose energy from the carbon skeletons, and excret-
their activity over time. The needs of many ing the nitrogen in the form of compounds
cells vary from minute to minute and cells of such as urea. Animals may also degrade pro-
all organisms adapt to this by changing, to teins to produce amino acids from which they
varying extents, the proteins which they make can make glucose if they cannot obtain suffi-
and hence the functions which they are able to cient carbohydrates from their diets or from
perform. stored glycogen (see Chapter 18).
There are other circumstances in which In contrast to animals, plants show indeter-
proteins must be broken down. Animals minate growth, that is they do not stop grow-
obtain amino acids in their diets in the form of ing when they reach a particular size. Often
proteins. However they cannot directly use their growth is limited by the amount of nitro-
dietary proteins of either plant or animal ori- gen available from the soil or the atmosphere
gin to make their own proteins. Instead almost and therefore they only rarely have to deal
all dietary proteins have to be broken down in with an excess of nitrogen. Thus amino acid
the gut before absorption as amino acids. oxidation occurs to a smaller extent than in
These amino acids then undergo processing to animals. Plants do, however, need to break
ensure that they are available in the propor- down amino acids produced as a result of pro-
tions required by the animal, which are often tein turnover or from the breakdown of stor-
not the same as those present in the diet. As age proteins, for example in seeds. Under
194 Breakdown of proteins and the oxidation of amino acids
these circumstances proteins are broken down protein ubiquitin (76 amino acids), by forma-
to amino acids which may be degraded and tion of covalent bonds between lysine residues
subsequently give rise to other amino acids in the proteins and the C-terminal COOH
according to the needs of the cells. group of the ubiquitin. The conjugates are
then rapidly degraded by the action of specif-
ic proteases. On the other hand, formation of
14.2 BREAKDOWN OF PROTEINS
complexes with molecules such as heat-shock
The first stage in the breakdown of proteins is proteins may stabilize proteins against attack
their hydrolysis to amino acids. There is a range by proteolytic enzymes.
of proteolytic enzymes which catalyse these The turnover of proteins in a 70 kg man
reactions. Some of these are relatively non-spe- result in release of about 400 g of amino acids
cific in their action, but others will only hydrol- per day, and up to 25% of these may be oxi-
yse bonds next to specific, individual amino dized or used to synthesize sugars.
acids within the protein sequence. Hydrolysis
of proteins takes place in the digestive system
14.3 BREAKDOWN OF AMINO ACIDS
and in cells during protein turnover.
In both ruminants and non-ruminants, the
amino acids released during digestion are
14.2.1 DIGESTION OF PROTEINS
absorbed in the small intestine and pass to the
Extensive degradation of proteins occurs dur- liver. The liver is the main site of amino acid
ing digestion. This process is discussed in metabolism but kidney also has some capacity
Chapter 28. to degrade amino acids. Some amino acids may
be metabolized in the liver or may pass to other
tissues where they are used to synthesize new
14.2.2 PROTEIN TURNOVER
proteins. Any excess amino acids are oxidized.
In all organisms, proteins are constantly syn- Amino acids produced as a result of protein
thesized from amino acids and then broken turnover also pass to the liver for processing.
down again. A proportion of the amino acids Although each of the 20 amino acids found
released are oxidized and completely degrad- in proteins is oxidized by a separate pathway,
ed. This may seem wasteful but it maintains a the first step in their oxidation is usually the
supply of active proteins and allows the removal of the amino group. This may occur
organism to adapt to changing conditions by either transamination or oxidative deami-
which it encounters. nation.
Not all proteins tum over at the same rate.
In general, structural or storage proteins tum 14.3.1 TRANSAMINATION REACTIONS
over more slowly than those involved in
Transamination involves removal of the a-
metabolism (Table 14.1). Proteins containing
regions rich in the amino acids proline, gluta- amino group of the amino acid and its transfer
mate, serine and threonine appear to have Table 14.1 Turnover of different classes of proteins
short half lives, and the presence of certain
amino acids at the amino terminus also Protein Half life
appears to accelerate breakdown. In addition, (days)
abnormal or damaged proteins also break
down rapidly, probably because they cannot Proteins of blood serum, liver and kidney 2-10
adopt the stable conformation of the normal Haemoglobin 30
Muscle proteins 180
protein. Once these proteins have been recog- Collagen 1000
nised they become conjugated to the small
Breakdown of amino acids 195
R, R2 R, R2
I I I I
CH-COOH + C-COOH C-COOH + CH-COOH
I II I
NH2 o o• NH2
a-amino acid 1 + a-ketoacid 2 ~ a-keto acid 1 + a-amino acid 2
a-amino acid + a-ketoglutarate ~ a-keto acid + glutamate

Figure 14.1 Transamination reactions take place between an amino acid and an a-keto acid. The amino
group is transferred to the ketoacid producing a new amino acid and a new ketoacid. The reactions are
readily reversed and are used in both breakdown and synthesis of amino acids. The amino group accep-
tor is often a-ketoglutarate which is converted to glutamate.
to an a-keto acid. The amino acid itself is con- net amino acid breakdown. Thus the process of
verted into the corresponding a-keto acid. The oxidative deamination is essential for release of
reaction is shown in Figure 14.1. Reactions of ammonia from amino acids. In this way the a-
this type are catalysed by transaminases, also amino group of the amino acid is converted
known as aminotransferases. directly to ammonia. The oxidative deamina-
Although a number of keto acids can take tion of glutamate, catalysed by glutamate
part in this reaction, a-ketoglutarate is often dehydrogenase, occurs very commonly (Figure
used and glutamate is produced (Figure 14.1). 14.2). This results in the production of ammo-
This reaction is catalysed by glutamate nia which is then converted rapidly into urea.
transaminase.
Transamination reactions are completely
14.3.3 OXIDATION OF CARBON SKELETONS OF
reversible and, as will be seen in Chapter 20,
AMINO ACIDS
can also be used to synthesize amino acids.
Many amino acids can take part in this reaction, Removal of the amino groups from amino
their amino groups are incorporated into gluta- acids by transamination or deamination
mate. Thus the nitrogen from the amino acids is results in the production of the corresponding
concentrated as the amino group of glutamate. a-keto acids. These can be further oxidized by
All transaminases function in a similar way a series of pathways, resulting in the produc-
and have pyridoxal phosphate as a tightly tion of metabolites which include pyruvate,
bound coenzyme. When the amino group is acetyl-CoA and the TCA-cycle intermediates.
removed from the amino acid it is initially They may be completely oxidized to carbon
transferred to the pyridoxal group bound to the dioxide, or in some cases, converted into sug-
enzyme (converting it to pyridoxamine phos- ars such as glucose by gluconeogenesis.
phate) but it is subsequently transferred to the In plants, the keto acids formed by break-
keto acid, regenerating pyridoxal phosphate. down of amino acids are used mainly in syn-
thesis of new amino acids and are only
degraded by oxidation to a very minor degree.
14.3.2 DEAMINATION
The pathways by which keto acids are bro-
Because transamination replaces one amino ken down are complex and will not be consid-
acid by another, it does not actually result in ered in detail in this text. However some
COOH COOH
I I
CH 2 CH 2
I I
CH 2 + NAD+ + H20 -- CH2 + NADH + H+
I I
CH-NH2 C=O
I I
COOH COOH
Glutamate a-ketoglutarate
Figure 14.2 Oxidative deamination of glutamate catalysed by glutamate dehydrogenase. Oxidation of
glutamate is accompanied by reduction of NAD+ to NADH and is irreversible.
important principles can be understood by breakdown. The end products of breakdown

considering the amino acids to belong to fam- of the carbon skeletons of amino acids are
ilies according to the end product of their shown in Figure 14.3.
Serine
Tryptophan
Alanine
Cysteine
_Threonine
.....
_ _ _- J ........ ,-..
L ~~oA+--
pyruvate Phenylalanine
Tyrosine
Isoleucine
Leucine
Lysine
Asparagine
-~"'O'~~\
Tryptophan
Arginine Threonine
Glutamate
Histidine
) Proline
-----,
fumarate -----'
, , Jf '- Succinyl-CoA
Phenylalanine
"- Succinate / ' ~"
Tyrosine "
Aspartate ,--"'_ _ _,,,
Isoleucine
Methionine
Valine
Threonine
Figure 14.3 Pathways for the oxidation of the carbon skeletons of amino acids to form TCA-cycle intermedi-
ates or related compounds. These may be further oxidized or used in biosynthetic reactions. Amino acids
which are converted into three- or four-carbon compounds can give rise to sugars and are glucogenic.
Breakdown of amino acids 197
Pyruvate or intermediates of the TCA cycle 14.3.5 THE UREA CYCLE
containing three or more carbon atoms can be
The urea cycle is the pathway by which urea is
converted into oxaloacetate and then into glu-
synthesized from ammonia and takes place
cose. Hence amino acids which are broken
mainly in the liver. Urea has two amino
down into these compounds are called gluco-
groups, one comes directly from ammonia and
genic amino acids. On the other hand, com-
the other from the amino group of aspartate.
pounds such as acetate (in the form of acetyl
The pathway for the synthesis of urea is a
CoA or acetoacetyl CoA, which itself breaks
cyclic one, and was largely discovered in 1932
down to acetyl CoA) do not result in oxalo-
by H.A. Krebs and his colleagues, the same
acetate production and therefore cannot be
people who elucidated the TCA cycle. The
used as precursors of glucose. Instead, the
pathway itself is shown in Figure 14.5.
acetyl CoA is converted into a range of other
Ammonia first reacts with carbon dioxide
compounds, including the ketone bodies.
and two molecules of ATP to produce a com-
Amino acids forming acetyl CoA are therefore
pound called carbamoyl phosphate. Only
called the ketogenic amino acids. A third
one of the ATPs is needed to provide the
group of amino acids break down into several
fragments, some of which are ketogenic and phosphate group, the other provides the
energy to drive the reaction. The next four
some glucogenic. These acids must be consid-
ered to be both glucogenic and ketogenic. intermediate compounds are all amino acids
but only one of them, arginine, is ever found
as part of proteins. Carbamoyl phosphate
14.3.4 THE FATE OF AMMONIA
reacts with the amino acid, ornithine, to pro-
Ammonia is very toxic and, in most organisms, duce another amino acid, citrulline. This then
any formed by oxidative deamination is rapid- reacts with a molecule of aspartate to pro-
ly converted into less-toxic organic com- duce argininosuccinate. This step requires a
pounds. Where water is plentiful, ammonia large amount of energy so that ATP has to be
may be excreted directly, as in protozoa, broken down to AMP and pyrophosphate
nematodes and aquatic amphibians. In most (PP), using the equivalent of two high-ener-
terrestrial animals ammonia is converted into gy phosphate bonds. The amino group of the
urea by the urea cycle, before being excreted. aspartate is derived from glutamate by
In birds and many terrestrial reptiles, nitrogen transamination.
is excreted predominantly as uric acid (Figure The arginine produced at this stage is then
14.4) which is synthesized from ammonia via hydrolysed, so that the end of the molecule is
the pathway which forms purines (see liberated as urea. This regenerates ornithine,
Chapter 21). with which the cycle began. Thus both of the
:x
o
HN )=
N
H
O~N N 0
H H
Uric acid
Urea
Figure 14.4 The structures of urea and uric acid, end products of nitrogen metabolism in most terrestrial
animals.
IX amino group of glutamate

.NH,T NH2
I
c=o
I
0
NH2 2ATP I
'O-P=O
I + CO2
C=O NH2 + H2O I
I O·
I
NH2 (CH 2h
I Carbamoyl
Urea CH-NH2 phosphate
~I
COOH
NH2 Ornithine
I
C=NH
I NH2
NH I
I C=O
(CH2h I
I NH
CH - NH2 I
I (CH 2h
COOH C02H I
CH - NH2
I
Arginine CH 2 I
COOH
I
\
ATP
CH-COOH
Citrulline
I
NH
I
C=NH
I C02H
NH I
I CH 2
(CH 2h I
+
I CH - NH2
CH - NH2 PPi
I
I COOH
COOH
Aspartate
Arginino-
succinate
IX Ketoglutarate
Oxaloacetate
Glutamate
Figure 14.5 The urea cycle takes place mainly in the liver and converts ammonia into urea. Four high.
energy phosphate bonds are broken for each turn of the cycle.
Precursor functions of amino acids 199
amino groups of the urea are ultimately Table 14.2 Examples of precursor functions of
derived from the amino group of glutamate. amino acids
The excretion of ammonia by way of urea
Amino acid Product
requires a considerable amount of energy.
Each turn of the cycle requires the expenditure Aspartate Pyrimidines
of four high-energy phosphate bonds (two Glycine Purines; glutathione (tripeptide: 'Y-
ATPs change to ADP and one changes to glutamyl cysteinyl glycine); por-
AMP). Urea is excreted through the kidneys in phyrins e.g. haem, chlorophyll
the urine. This means that there is also a high Histidine Histamine - a biologically active
requirement for water in the process. As the amine released from some animal
tissues in response to inflammation
main end product of their amino acid metabo-
or as an allergic reaction causing
lism, birds and reptiles excrete uric acid in the dilation of blood vessels
form of a solid suspension which reduces the Glutamic acid 'Y-aminobutyric acid (GABA) -
need for water in their excretory processes. neurotransmitter in brain
In addition to urea, mammals also excrete Tyrosine Adrenalin; morphine; thyroid hor-
small amounts of uric acid, not as an end prod- mone
uct of amino acid catabolism, but formed by Tryptophan Nicotinic acid (component of NAD+
etc.); serotonin (neurotransmitter);
the breakdown of purines.
indole acetic acid (plant hormone)
Valine Pantothenic acid (component of
14.3.6 THE FATE OF UREA IN RUMINANTS coenzyme A)
In ruminant animals some of the urea that is

produced in the liver is not captured by the A common reaction is the decarboxylation
kidneys but instead may be returned to the of amino acids to form amines, e.g. histamine
rumen in saliva or directly through the rumen and 'Y-aminobutyric acid. These reactions are
wall. Microbes in the rumen can use the urea catalysed by pyridoxal-requiring decarboxy-
to make their own cell proteins. The microbial lase enzymes. In the case of many alkaloids,
cells then pass further down the digestive further oxidation of amines to aldehydes and
tract to the abomasum (true stomach) and condensation of these with amines gives rise
small intestine where the proteins that they to the heterocyclic rings which are found in
contain are digested. In this way ruminants these compounds.
reduce their dependence on external supplies
of protein.
14.4 PRECURSOR FUNCTIONS OF AMINO

ACIDS
As well as being constituents of proteins,
many amino acids are also precursors of other
important compounds. Some of these func-
tions are indicated in Table 14.2.
THE PENTOSE PHOSPHATE PATHWAY 15

15.2 Oxidative reactions 201
15.3 Rearrangement reactions 201
15.4 Importance of the pathway 205
15.5 Regulation of the pathway 206
15.1 INTRODUCTION erating some glucose-6-phosphate from the

ribulose-5-phosphate produced in the oxida-
The pentose phosphate pathway (also known
tion steps (Figure 15.3).
as the phosphogluconate pathway or the hex-
Ribulose-5-phosphate is converted into
ose monophosphate shunt) provides a route
other pentose sugars: ribose-5-phosphate by
by which glucose can be oxidized to carbon
the action of ribulose phosphate isomerase [5],
dioxide. As in the case of glycolysis, the sub-
strate for the pathway is glucose-6-phosphate. and xylulose-5-phosphate by the action of ribu-
An outline of the pathway is shown in Figure lose phosphate-3-epimerase [4] (Figure 15.1).
These sugars are then acted on by transketolase
15.1. The pathway can be considered in two
parts: oxidative reactions and rearrangement [6] (Figures 15.1 and 15.3) which transfers a
reactions. -CO-CHzOH group from the ketose sugar
(xylulose-5-phosphate) to the aldose (ribose-5-
phosphate). This reaction results in the produc-
15.2 OXIDATIVE REACTIONS tion of a seven-carbon sugar (sedoheptulose-7-
In these reactions, glucose-6-phosphate is oxi- phosphate) and a three-carbon one (glyceralde-
dized by successive removal of two pairs of hyde-3-phosphate). These two sugars then
hydrogen atoms. The first step, catalysed by the react together in a step catalysed by transal-
enzyme glucose-6-phosphate dehydrogenase dolase [7] (Figures 15.1 and 15.3) in which a
[1], forms 6-phosphogluconolactone (Figure three-carbon fragment (-CHOH-CO-CHzOH)
15.2). This then undergoes hydrolysis, catalysed is transferred from sedoheptulose-7-phosphate
by lactonase [2], producing 6-phosphoglu- to the glyceraldehyde-3-phosphate, resulting in
conate as a result of ring opening. Oxidative six-carbon (fructose-6-phosphate) and four-
decarboxylation of 6-phosphogluconate, catal- carbon (erythrose-4-phosphate) sugars. Trans-
ysed by 6-phosphogluconate dehydrogenase ket-olase is not specific in the acceptor it uses for
[3], yields the five-carbon sugar ribulose-5- its transfer reaction, and also catalyses transfer
phosphate. In both oxidation steps, hydrogen of a two-carbon fragment from xylulose-5-
atoms removed from the substrates are trans- phosphate to erythrose-4-phosphate, forming
ferred to NADP+, reducing it to NADPH. six- and three-carbon products (fructose-6-
phosphate and glyceraldehyde-3-phosphate,
respectively). Condensation of three-carbon
15.3 REARRANGEMENT REACTIONS
fragments, catalysed by triose phosphate iso-
The remainder of the pathway consists of a merase [8], aldolase [9] and phosphoglucose
series of reactions in which sugars undergo isomerase [10], results in eventual production
rearrangement and transfer reactions, regen- of glucose-6-phosphate.
202 The pentose phosphate pathway
Ribulose-5-
phosphate ...--c:::::::---------=-------
(6 x C s) ~ [~ \41
cO2 ~ 6NADPH + 6H+ \ \
[3] ~ 6NADP+ Ribose-5- Xylulose-5-

phosphate phosphate
6-phospho- (2 x Cs) (2 x Cs)
gluconate
(6 x C s)
Sedoheptulose- Glyceraldehye-
7-phosphate 3-phosphate
(2 x C7 ) (2 x C 3)
6-phospho-
gluconolactone
(6 x C s)
~
6NADPH + 6H+ Fructose-6- Erythrose-4-
[11 phosphate phosphate
6NADP+ Xylulose-5-
(2 x C6 ) (2 x C4 ) phosphate
(2 x C s)
Glucose-6-
phosphate
(6 x C 6 )
r
Fructose-6- Glyceraldehye-
phosphate 3-phosphate
(2 x C6 ) (2 x C 3)
Glucose-6-
phosphate
(5 x C6 )
Glyceraldehye-
3-phosphate
(1 x C3)
[8]
Fructose-6-
phosphate
(5 x Cs) [9]
Dihydroxy-
acetone
phosphate
(1 x C 3)
Figure 15.1 The pentose phosphate pathway. Enzymes catalysing individual steps are as follows: [1] glu-
cose-6-phosphate dehydrogenase, [2]lactonase, [3] 6-phosphogluconate dehydrogenase, [4] ribulose-
phosphate-3-epimerase, [5] ribulose-phosphate isomerase, [6] transketolase, [7] transaldolase, [8]
triosephosphate isomerase, [9] aldolase, [10] phosphoglucose isomerase.
Rearrangement reactions 203
vt-0\J
CH 2o@
H~OH Glucose-6-phosphate
H OH
~
NADP+
[1)
NADPH + H+
CH 2o@
o
H H
HO OH
H
H
OH
0
6-Phosphoglucono-
lactone
C0 2 H
H-cLoH
Ho-cLH CH 20H
H-~-OH ~-O
6-Phosphogluconate
H-~-OH HO-~-H
I
CH 2 o@
H-~-OH
I
K
CH 2o@
NADP+
[3)
Xylulose-5-phosphate
NADPH + H++ CO2
CHO
CH 20H
~-O H-~-OH
H-~-OH H-~-OH
H-~-OH H-~-OH
I
I CH 2o@
CH 2o@ [5]
Ribulose-5-phosphate Ribose-5-phosphate
Figure 15.2 Oxidative reactions of the pentose phosphate pathway. Both oxidation steps produce
NADPH. The names of the enzymes are given in the legend to Figure 15.1.
CH 20H
CH 20H CHO t-o

t-o H-t-OH [6'] H0-6-H CHO
HO-t-H + H-6-0H ....--
~
H-6-0H
+ H-6-0H
I
H-6-0H H-6-0H H-6-0H CH 2o@)
I I
CH 2o@) CH 2o@) H-t-OH
I
CH 2o@)
Xylulose-5- Ribose-5- Sedoheptulose-7- Glyceraldehyde-3-

phosphate phosphate phosphate phosphate
CH 20H
t-o CHO
CH 20H
t-o
H0-6-H CHO [7]
H-6-0H H0-6-H
H-6-0H
+ H-6-0H ....--
~
+
I H-t-OH H-6-0H
H-6-0H
CH 2o@) I
CH 2o@) H-6-0H
H-6-0H I
I CH 2o@)
CH 2o@)
Sedoheptulose-7- Glyceraldehyde-3- Erythrose-4- Fructose-6-

CH 20H
CH 20H
CHO 6-0
6-0 CHO
H-6-0H
[6']
H0-6-H
H0-6-H + ....--
~
H-6-0H +
H-6-0H I H-6-0H
H-6-0H
I
I CH 2o@)
CH 2o@) H-6-0H
CH 2o@) I
CH 2o@)
Xylulose-5- Erythrose-4- Glyceraldehyde-3- Fructose-6-

Figure 15.3 Rearrangement reactions of the pentose phosphate pathway. These reactions are reversible
and allow the interconversion of sugars containing 3-, 4-, 5-, 6- and 7-carbon atoms. The names of the
enzymes are given in the legend to Figure 15.1.
Importance of pathway 205
The overall equation for this pathway is as therefore serve as a source of ribose, required
follows: for the production of nucleotides and nucleic
acids, and of erythrose-4-phosphate, a precur-
Glucose-6-phosphate + 12 NADP+ + 7H2 0 --->
sor of the aromatic rings which form part of
6C02 + 12NADPH + 12H+ + Pi
some amino acids and phenolics, such as
lignin, tannins and anthocyanins (Chapter 5).
The latter process does not occur in animals
15.4 IMPORTANCE OF THE PATHWAY
which are unable to synthesize aromatic com-
Although the pentose phosphate pathway pounds.
and glycolysislTCA cycle both bring about The pentose phosphate pathway has sever-
complete oxidation of glucose to carbon diox- al intermediates in common with glycolysis.
ide, they serve different purposes. The pen- The rearrangement steps of the pathway are
tose phosphate pathway produces NADPH, readily reversible and can be used to make
whilst the glycolysis/TCA cycle produces ribose and erythrose from glucose-6-phos-
NADH. As has been noted elsewhere (Chapter phate. The pentose phosphate pathway and
6), NADPH is used primarily as a reducing glycolysis, operating together, provide a very
agent in biosynthetic reactions, whilst NADH versatile system which can work in several dif-
serves as a source of hydrogen atoms for the ferent ways, depending on the relative
electron transport chain. Thus, whilst glycoly- requirements of the cell for NADPH and
sis is mainly concerned with energy metabo- ribose-5-phosphate.
lism and is active in all tissues, the pentose
• Much more NADPH than ribose-5-phos-
phosphate pathway is found mainly in tissues
phate required: the complete pathway,
in which biosynthetic processes are important.
using both oxidative and rearrangement
NADPH is required in large amounts for the
reactions, leads to oxidation of glucose-6-
synthesis of fatty acids and steroids, and is also
phosphate to carbon dioxide.
required for production of deoxyribonu-
• Much more ribose-5-phosphate than
cleotides. The pathway is therefore active in
NADPH required: glucose-6-phosphate is
liver, lactating mammary gland and adipose
converted to fructose-6-phosphate and
tissue. In plants, although NADPH is pro-
glyceraldehyde-3-phosphate by glycolysis,
duced in the chloroplasts during the light
and then ribose-5-phosphate is produced
reactions, it is not readily available to the cyto-
by reversal of the rearrangement steps. The
plasm and is, in any event, only produced in
oxidative steps of the pathway are not used.
this way in photosynthetic tissues. Thus the
This situation arises, for example, in rapidly
pentose phosphate pathway is also required
dividing cells.
by plants as a source of NADPH and of ribose
• Balanced requirement for NADPH and
and erythrose-4-phosphate.
ribose-5-phosphate: the oxidative steps are
A further function of NADPH may be to
used to produce two NADPH molecules for
keep glutathione in its reduced form. In ani-
each ribose-5-phosphate. Rearrangement
mals, deficiencies of the enzyme glucose-6-
steps are not required.
phosphate dehydrogenase lead to lowered
levels of reduced glutathione, so making red The pathway can provide a mechanism by
blood cells susceptible to damage from hydro- which pentose sugars are utilized. These may
gen peroxide. be present in animal diets as components of
The pentose phosphate pathway has, as hemicellulose, xylans, pectins and nucleic
intermediates, sugars containing three-, four-, acids. In this way, pentose sugars can be used
five-, six- and seven-carbon atoms. It can for biosynthesis or energy production.
15.5 REGULATION OF THE PATHWAY NADPH. If NADPH is used to synthesize fatty
acids, the activity of the enzyme, and hence
In view of the variety of ways in which the
production of NADPH, increase. On the other
pathway can operate, it is not surprising that it
hand, accumulation of fatty acyl CoAs
is subject to tight regulation. The enzyme glu-
decreases the activity of the enzyme and
cose-6-phosphate dehydrogenase is the main
NADPH synthesis.
point of control. This enzyme is inhibited by
FERMENTATION PATHWAYS 16
16.2 Anaerobic environments in agriculture 207
16.3 Lactate production 207
16.3.1 Muscle metabolism 208
16.3.2 Regeneration of glucose 208
16.4 Animal digestive systems 208
16.4.1 Acetate formation 209
16.4.2 Propionate formation 209
16.4.3 Butyrate synthesis 210
16.5 Soils 210
16.6 Waste treatment 211
16.7 Methane production 213
16.8 Dairy products 213
16.9 Meat 213
16.10 Fermentation in herbages 214
16.11 Ethanol production 214
16.1 INTRODUCTION environment precludes the penetration of

It has been assumed in previous chapters that
oxygen, as for example in:
there is an abundant supply of oxygen and • muscle contraction when demand for oxy-
that most biological oxidations can be thought gen is greater than the supply from the cir-
of as controlled versions of combustion. There culation
are a number of situations in which oxygen • animal digestive systems
supply may be very limited or even non-exis- • soils
tent, but many types of cell survive under • waste treatment
these conditions. In biological systems, oxida- • fermentation in milk products
tion normally takes place by the addition of • changes in meat post mortem
oxygen, the removal of hydrogen or the • changes in crops after harvest
removal of electrons. Hydrogen atoms that are • ensiling of forages
removed are subsequently oxidized to water • production of ethanol.
under aerobic conditions. Even under anaero-
bic conditions, oxidation is made possible by
16.3 LACTATE PRODUCTION
the movement of electrons.
The biochemical pathway for lactate produc-
16.2 ANAEROBIC ENVIRONMENTS IN
tion makes use of glycolysis, in which glucose
AGRICULTURE
is broken down to pyruvate. This pyruvate is
then utilized for lactate synthesis. For each
Anaerobic conditions can develop even in sup- mole of glucose converted to pyruvate, glycol-
posedly aerobic organisms such as animals, ysis produces four moles of ATP and two of
but principally they are to be found where the NADH, and consumes two moles each of
208 Fermentation pathways
NAD+ and ATP (Chapter 10). In terms of use- Slow-twitch cells obtain most of their energy
ful biochemical energy, the yield is two moles directly from the aerobic oxidation processes
of ATP from each mole of glucose. This would of the mitochondria with which they are well
be a useful way of producing a limited amount supplied. The fast-twitch cells can also obtain
of energy from glucose in the absence of oxy- energy from the anaerobic oxidation of glu-
gen, but cells would soon run out of NAD+ cose (released from intracellular glycogen
and the process would grind to a halt. This stores) to lactate.
problem is overcome by changing the pyru-
vate into lactate (lactic acid), which changes
16.3.2 REGENERATION OF GLUCOSE
the two molecules of NADH back into NAD+
(Figure 16.1). The reaction, which is easily The lactate produced in muscle is passed via
reversed, is catalysed by the enzyme lactate the blood to the liver where it is used as a pre-
dehydrogenase. In this way the overall path- cursor of glucose synthesis (see Chapter 18).
way from glucose to lactate can continue to The glucose synthesized in the liver can then,
catabolize glucose and gain a small amount of in turn, be sent to back to the muscle. This
energy in the form of the two molecules of cycle of reactions has been named the Cori
ATP. This is only one of two schemes for lac- cycle after its discoverers C.F. Cori and G.T.
tate production from glucose; it differs from Cori (see Figure 16.2).
the other in producing only lactate as its prod- If lactate starts to accumulate due to sus-
uct, and it is therefore known as a homolactate tained physical activity, then it is a common
fermentation. The yield of ATP from this path- experience in man that the muscles ache - the
way is low, but the substrate has not been actual pain may be due to the decrease in intra-
extensively degraded and the product, lactate, muscular pH caused by the lactate produced.
is available for other purposes.
16.4 ANIMAL DIGESTIVE SYSTEMS
16.3.1 MUSCLE METABOLISM
Anaerobic conditions are found in parts of the
Muscles require large amounts of energy to gut in all animals. It has only recently been
function, but the work performed varies. appreciated that the anaerobic fermentation in
Some need to be able to respond very quickly the human large intestine may have an impor-
to changing situations, whereas others may be tance of its own. However, fermentation is
required to maintain a constant work output essential in herbivores, in the rumen and retic-
but at a relatively low intensity. Within mus- ulum of ruminants and tylopods (camels etc.),
cles a number of different types of fibre have or in the caecum of the non-ruminant herbi-
been identified which lie in a spectrum from vores (rabbits, horses etc.). The major end
fast-twitch to slow-twitch: these are often products of these fermentations are the
denoted as white and red fibres, respectively. volatile fatty acids which supply most of the
animals' dietary energy. In non-herbivores
COOH COOH
I I this role is played by glucose. There are three
c=o HO-C-H major volatile fatty acids: acetate, propionate
I I
CH 3 CH 3 and butyrate, together with a number of
Pyruvate
Lactate
dehydrogenase Lactate
minor ones. They are produced by microor-
ganisms - bacteria, fungi and protozoa which
Figure 16.1 Reduction of pyruvate to lactate.
Together with the glycolytic pathway, this reaction
live symbiotically in the digestive tract. The
leads to the production of lactate from glucose; lac- microorganisms benefit by gaining their ener-
tate is the only product and thus the process is gy from the partial breakdown of the carbohy-
known the homolactate pathway. drates eaten by the animals. The end products
Animal digestive systems 209
include many (e.g. cellulose and hemicellu-
lose) that cannot be broken down by simple-
stomached animals such as man. The microbes
are also able to utilize simple sources of nitro-
gen as precursors for the synthesis of amino
acids and proteins. In ruminants, these pro-
teins eventually become available to the ani-
mal as they pass further through the digestive
tract. Additional benefits are brought to the
Glucose
Lactate MUSCLE animal in the form of a supply of vitamins.
The common feature of the pathways for
volatile fatty acid synthesis from glucose is
that they all involve pyruvate.
16.4.1 ACETATE FORMATION
The simplest route is the pathway for acetate

production, in which the pyruvate reacts with
coenzyme A forming acetyl-CoA and losing
formate (Figure 16.3). The acetyl-CoA loses its
coenzyme A with the simultaneous produc-
tion of ATP, part of the benefit accruing to the
microbe in the form of energy. The formate is
split into carbon dioxide and gaseous hydro-
gen. Hydrogen is removed by conversion to
methane.
Lactate Glucose 16.4.2 PROPIONATE FORMATION

Propionate is the only one of the three major
volatile fatty acids that the ruminant can use
as a source of glucose. There are two different
pathways for propionate synthesis from pyru-
vate, each favoured by different types of
microbes (Figure 16.4). One of these pathways
proceeds by an extension of the route for the
synthesis of lactate. Lactate is first activated by
Figure 16.2 The Cori cycle. Glucose passes from
the liver to muscle where it is oxidized to lactate. the formation of its coenzyme A ester. This
The lactate is then returned to the liver where it is loses both CO 2 and water to form propernoyl-
used for glucose synthesis. Under conditions of CoA, the unsaturated analogue of propionyl-
high energy demand, such as exercise, lactate may CoA. In turn this is hydrogenated to yield pro-
temporarily accumulate in muscle. pionyl-CoA, which loses coenzyme A to yield
the product. The second pathway for the gen-
eration of propionate is virtually a reversal of
of microbial metabolism are available to the the route that is used in animal cells, and par-
host animal, which gains because the carbohy- ticularly the liver, to synthesize carbohydrates
drates that are fermented by microorganisms from propionate (see Chapter 18).
COOH
I Pyruvate
C=O
I
CHs
CoASH
SCoA
I
Acetyl CoA C = 0
I
CHs H-COOH Formate
ADP+ PI~
ATP~COASH
COOH
I
CHs
Acetate
Figure 16.3 The fermentation of glucose to produce acetate. Formate produced in the decarboxylation of
pyruvate is broken down to CO 2 and gaseous hydrogen.
16.4.3 BUTYRATE SYNTHESIS yield the coenzyme A ester of butyric acid. The
The first three steps of butyrate production are ester is hydrolysed to produce butyrate and
free coenzyme A. This reaction is coupled to
reactions that are commonly encountered
the phosphorylation of ADP to ATP, which
under other circumstances (Figure 16.5). The
provides more energy for the metabolism of
first one is the pyruvate dehydrogenase step,
which links glycolysis with the tricarboxylic the microorganism.
acid cycle. As a start to the production of
butyrate, two molecules of pyruvate must be
16.5 SOILS
oxidized to yield two molecules of acetyl-CoA.
Carbon dioxide is lost and NAD+ is reduced to Soils provide a mixture of aerobic and anaero-
NADH. Acetoacetyl-CoA is formed from two bic environments; which of these predomi-
molecules of acetyl-CoA , with one molecule of nates at one time depends on the porosity of
coenzyme A being liberated. Acetoacetyl-CoA the soil, and on the extent to which air move-
is reduced to yield 3-hydroxybutyryl-CoA, uti- ment is impeded by close packing of the soil
lizing NADH and liberating NAD+. matrix or by the filling of the pores by water.
The hydroxybutyryl-CoA then loses water to Much of the decomposition of organic materi-
form an unsaturated compound, crotonyl-CoA. al in soils takes place through aerobic oxida-
Similar, but not identical, steps are to be found tion; even 02 concentrations of as little as 1 %
both in fatty acid oxidation and synthesis. may be sufficient to ensure aerobic degrada-
NADH produced in earlier parts of the tion. It is for this reason that in many soils the
pathway is used to reduce crotonyl-CoA to organic matter does not reach very high levels
Waste treatment 211
GTP
co Oxalacetar\e SUCCina~e
2~ GOP
+ P.
CoASH I
IPyruvatel NADH + H+
FADH2
NAD+
FAD
0
~
CSCoA
~
ADH + H+
Succlnyl CoA yH2
I
NAD+ CH 2
I
COOH
Lactate
0 ...
"CSCoA
1
Lactyl CoA
H.t~C:~ MethYlm~alonYI CoA
CO, + H,01 Acetyl CoA

o Propenoyl CoA 0... cO2
~C SCoA
f:
'bI SCoA CH 2
I
Proplonyl CoA
CH Acetate I
II COOH
CH 2 AMP
+ P~
NADH + H+
CoASH ATP
IPropionate I
Propionyl CoA
0 ...
0 ... ~C·OH
~C SCoA I
I CH 2
CH2 I
I COOH
CH3
Figure 16.4 The two pathways for the fermentation of glucose to propionate. One pathway passes
through lactate and involves a complex 'shuttle' of Coenzyme A via acetyl-CoA. The other pathway, via
succinate, is the reverse of the mechanism used by animals to synthesize glucose from propionate.
despite large inputs of decaying vegetation gen sulphide, a common product in anaerobic
each year. There are systems where decay is biological systems.
severely impeded and anaerobic conditions
prevail. The enormous deposits in the peat
16.6 WASTE TREATMENT
bogs in cool temperate regions are a testimony
to the effect of anaerobic fermentation. In the One of the features of the intensification of agri-
absence of oxygen, anaerobic organisms can culture in the 20th century has been an increas-
use a whole range of other compounds as ing awareness of the problems associated with
receptors for electrons. A good example is the agricultural wastes - particularly where these
use of sulphur compounds which are reduced are produced close to centres of human popu-
to the sulphide. Under acid conditions this lation. Basically there are two types of waste
associates with hydrogen ions to form hydro- treatment, aerobic and anaerobic. In the former
+
NAD+ NADH + H
COOH ~ SCoA
I
C =0 ? =0
I
2 Acetyl CoA
6H3
2 Pyruvate
2 CoASH
CO2
o
h
CH3
CoASH
"C- SCoA
I
Acetoacetyl CoA ?H2
c=o
I
K
CH 3
NADH + H+
o NAD+
"C- SCoA
3-hydroxybutyryl CoA I
CH 2
I
HO-CH
I
CH 3
}..H 0 2
o
"C- SCoA
I
CH
Crotonyl CoA II
HC
I
K
CH 3
NADH + H+
Butyrate
NAD+
o
"C-OH
I
Butyryl CoA CH 2
I
CH 2
I
CH 3
CoASH
Figure 16.5 The fermentation of glucose to yield butyrate. Although the first step produces NADH, there
are two susbsequent steps that absorb it. This allows the pathway to 'soak up' the NADH that is pro-
duced in glycolysis.
case, organic wastes are almost completely oxi- into the agricultural system. A product that is of
dized, with the eventual production of CO 2 and growing interest, particularly in the developing
water. Anaerobic oxidation yields a consider- world, is the production of methane (biogas) by
able amount of biomass which may be recycled anaerobic fermentation.
Meat 213
16.7 METHANE PRODUCTION Many anaerobic pathways in the rumen
produce hydrogen as an end product which
The reactions of methane synthesis are not
may be used for the hydrogenation of fatty
confined to biogas generation: they occur to a
acids in the rumen.
great extent in the digestive tracts of animals,
particularly ruminants. Four major precursors
are shown in Figure 16.6. In methane genera- 16.8 DAIRY PRODUCTS
tion from waste the first of these, using
acetate, tends to account for the larger part of Refrigeration is a comparatively recent tech-
gas production. nique for preserving dairy products, but the
In the rumen, the reactions that use hydro- use of anaerobic fermentation to yield aci-
gen, formate or methanol are the most impor- dophilic milk, yoghurt and cheese goes back
tant. Methane represents a fairly inert material to the dawn of civilization. Most of the initial
that overcomes the potentially toxic effects of fermentative changes in milk involve the
high concentrations of any of these precursors. homolactate pathway described for muscle.
On average, about 8% of the energy in diets
eaten by ruminants is released in the form of
16.9 MEAT
methane. This has led to concern that animals
may make a major contribution to the green-
I Once an animal is dead the supply of oxygen
house' gases thought to be responsible for to the muscles ceases abruptly. Anaerobic
global warming. catabolism then takes place in the tissues and
1. From acetate.
2. From Hydrogen and carbon dioxide
3. From Formate
4 H-COOH
4. From Methanol.
4 C~OH
Figure 16.6 Four different pathways for the synthesis of methane. The pathway used varies from
microorganism to microorganism.
to a great extent determines the quality of the There are many pathways to be found in fer-
meat product. Glycogen, present in the mus- menting herbage but the most important ones
cle at death, is oxidized anaerobically, leading are those that produce lactate. In general, good
to a build-up of lactate in the muscle which silages are those in which the commonest acid
reduces the pH within the tissue from its nor- is lactic acid and the levels of the volatile fatty
mal (living) value of about 7.3 to around S.4. acids are very low. One pathway for lactate
If animals are stressed in some way prior to production (the homolactate pathway) has
slaughter, the level of glycogen in their mus- been considered already. Another (the hetero-
cles is reduced. For instance, in a chicken that lactate pathway) takes place in silage (Figure
is struggling at the time of death, glycogen 16.7). This involves the prior breakdown of hex-
values may only be half of those for a ose sugars to pentoses (ribulose-S-phosphate
stunned animal. In turn, lactate production is and xylulose-S-phosphate); these steps are also
reduced and the pH values obtained are found in the first part of the pentose phosphate
higher, perhaps as high as 6.5. The character pathway. Xylulose-S-phosphate is cleaved into
of the meat is different: it is drier, darker and two compounds, one with three carbons and
more firmly textured than normal. In the beef the other with two. The three-carbon glycer-
industry this dark, firm, dry (OFO) meat can aldehyde-3-phosphate is metabolized via pyru-
cause significant financial losses (see Chapter vate (as in glycolysis) to lactate. The two-carbon
32). acetyl phosphate is merely dephosphorylated
(producing ATP) to give acetate.
The main objective in making silage is to
16.10 FERMENTATION IN HERBAGES
reduce the pH to a low value, preferably in the
In most parts of the world the production of region of 4.5, as quickly as possible. In the
fodder occurs on a seasonal basis. This may be presence of oxygen, the fermentation path-
because in winter the temperatures are too ways move towards the production of large
low for crop growth (as in temperate regions) quantities of butyric acid, which leads to an
or because water is in short supply during a unpleasant-smelling product with a pH of S.5
dry season (many tropical areas). Fodder crops or higher. Under these conditions the nutritive
from the growing season are preserved for use value of the product is very low and animals
during the shortage. One method is drying, are extremely reluctant to eat it. All effective
but this presents technical problems in many silage-making procedures depend on exclud-
environments. A large proportion of farmers ing air by compressing the herbage and seal-
now effectively pickle their crop by allowing it ing the silage carefully with an impermeable
to ferment. The acids which are produced material such as plastic sheet.
lower the pH to such an extent that a well pre-
served and palatable product is formed.
16.11 ETHANOL PRODUCTION
Almost all of the methods for silage-making
rely on anaerobic fermentation of carbohy- Some crops are grown specifically as the raw
drates (sugars or starch), these may be present material for alcohol fermentation. They all
in the crop as in temperate grasses or in maize, produce large quantities of sugars (e.g. sugar-
or they may have to be added, possibly as cane) or starch (grains and some roots).
molasses, in order to ferment the tropical The biochemical pathway is very simple: in
grasses. Whatever the source of carbohydrate essence it is an alternative use for the pyruvate
the processes are quite similar. The most produced during glycolysis. There are only
important condition that must be achieved is two extra steps (Figure 16.8). In the first step,
an absence of oxygen to ensure anaerobic fer- pyruvate loses CO 2 (decarboxylation) to yield
mentation (see Chapter 24). acetaldehyde. The reaction occurs irreversibly
Ethanol production 215
Glucose Arabinose Xylose
J
I
\+[2H]
co
t\ [2H]
Ribulose
/:; AlP
2 Ribulose- ADP Xylulose
S-Ph\,ate ~ATP
CHzOH ADP
bI=0 Xylulose-
HO-CH 5-phosphate
I
HC·OH
I
P;
cHzo® Phosphoketo/ase
HC= 0
I
HC·OH
tHzo<P> Glyceraldehyde- Acetyl
3-phosphate phosphate
ADP ,~ NAO+
AlP~I NADH + W ~ADP
1,3-bisphosphoglycerate
l~AlP
AD~I Acetate
AlP
Pyruvate
V NADH + H+
~NAD+
Lactate
Figure 16.7 The heterolactate pathway which operates during ensiling forages. This differs from the
homolactate pathway of Figure 16.1 in that a mixture of products, lactate and acetate, is formed.
under the influence of pyruvate decarboxy- The reducing power comes from NADH pro-
lase. This enzyme shares with the pyruvate duced in the earlier stages of glycolysis. The
dehydrogenase complex (Chapter 11) a NAD+ produced in this pathway can then be
requirement for thiamin pyrophosphate (TPP) recycled to glycolysis, allowing it to continue.
as a coenzyme. The pyruvate is attached to the Mammalian systems may sometimes have
molecule of TPP, whilst the decarboxylation is to metabolize ethanol which has either been
taking place. directly administered or produced by fermen-
The second step is reversible and involves tation in the gut of herbivores. The first stage
the reduction of acetaldehyde to yield ethanol. reverses the alcohol dehydrogenase reaction
1
NADH + H+ NAD+
COOH
V
H H
I I
c=o
I
'cI = 0 H-C-OH
I
CH 3 Pyruvate CH 3 Alcohol CH 3
decarboxylue dehydrogenase
pyruvate Acetaldehyde Ethanol
Figure 16.8 Reactions leading to the synt!lesis of ethanol from pyruvate.
to produce acetaldehyde. An aldehyde dehy- acetate, which can be further metabolized the
drogenase then uses NAD+ as a hydrogen through the TCA cycle or as a precursor for
acceptor to oxidize the acetaldehyde to fatty acid synthesis.
PHOTOSYNTHESIS 17
17.2 Chloroplasts 217
17.3 The Light reactions 217
17.3.1 Photosynthetic pigments 217
17.3.2 Light absorption 219
17.3.3 Resonance energy transfer 221
17.3.4 The electron transport system 221
17.3.5 Photo system I 222
17.3.6 Photo system II 223
17.3.7 Cytochrome b-f complex 225
17.3.8 Plastocyanin 225
17.4 Integration of the electron transfer system 225
17.5 ATP production 225
17.6 The dark reactions (Calvin cycle) 226
17.7 Control of photosynthesis 229
17.8 Photorespiration 229
17.8.1 Factors affecting rates of 230
photorespiration
17.9 Photosynthesis in C4 plants 232
17.10 Crassulacean acid metabolism 233
17.11 Herbicides and photosynthesis 234
17.1 INTRODUCTION It is generally considered that about two-

thirds of carbon dioxide fixation takes place on
Photosynthesis is the process in which the land and about one-third in the oceans, but
energy of light is used to bring about synthesis the contribution made by the latter has proba-
of complex organic molecules, such as sugars, bly been underestimated. The quantity of car-
from carbon dioxide and water. It is of enor- bon dioxide directly available to plants from
mous importance as it is the principal means the atmosphere makes up only about 0.001 %
by which energy can enter biological systems. of the total carbon on earth. Over 99% exists in
Photosynthesis taking place in the past pro- the form of rocks and sediments. About 13% of
duced the fossil fuels which form most of our the carbon dioxide in the atmosphere is used
current energy reserves. in photosynthesis and released by respiration
The scale on which photosynthesis occurs is each year, and an approximately equal quanti-
staggering. The amount of carbon fixed per ty exchanges with carbon dioxide dissolved in
year has been estimated to be of the order of the oceans.
100 billion tons (1 X 1011 tons) - equivalent to Photosynthesis occurs in a wide range of
about 200 billion tons of organic matter as organisms, from green plants and algae to
CHzO. Even temperate grassland, with its some bacteria. The processes themselves are
modest growth rates, may produce 600 g of always associated with biological membranes
organic matter per mZ per year. in which pigments are embedded. In eukary-
218 Photosynthesis
otes such as green plants and green algae, regions of the grana. It is in these areas that PS
these membranes are found in organelles II is mainly located. The parts of the grana at
called chloroplasts, but in prokaryotes such as the top and bottom of the stacks are called the
photosynthetic bacteria and blue-green algae, non-appressed regions. PS I and ATP synthase
the pigments are present in specialized mem- are restricted to these regions and to the stro-
branes within the cytoplasm. ma thylakoids, both of which are in close con-
The process of photosynthesis can be divid- tact with the stroma. The thylakoid mem-
ed into two parts. branes enclose a continuous cavity, the thy-
lakoid space, which they therefore separate
• Light reactions, in which light is absorbed
from the stroma.
and its energy used to make ATP and
NADPH from ADP and NADP+, respective-
ly. As described in more detail later in this 17.3 THE LIGHT REACTIONS
chapter, the light reactions are brought
Pigment molecules are responsible for absorb-
about by the flow of electrons through two
ing light and form part of discrete structural
photosystems: photosystem I (PS I) and
components of the membranes called photo-
photosystem II (PS II). This electron flow
systems, which are embedded in the thylakoid
generates a proton gradient across the mem-
membranes.
branes and this in turn drives the synthesis
of ATP by an enzyme called ATP synthase .
• Dark reactions or light-independent reac- 17.3.1 PHOTOSYNTHETIC PIGMENTS
tions, in which ATP and NADPH are used
Chlorophylls
to reduce carbon dioxide to produce sugars.
These reactions are brought about by the The chlorophylls are the main light-absorbing
operation of a metabolic pathway usually pigments. They contain a magnesium atom
known as the Calvin cycle. chelated by the pyrrole nitrogen atoms of a
porphyrin ring. Porphyrins are formed from
succinyl-CoA (an intermediate in the TCA
17.2 CHLOROPLASTS
cycle) and glycine (an amino acid) via an inter-
Chloroplasts are disc-shaped organelles about mediate called o-aminolevulinic acid (ALA)
5 J.Lm long. They are surrounded by a double- which gives rise to each of the four pyrrole
membrane system (Figure 17.1). The outer rings. One of the COOH groups of chlorophyll
membrane is relatively permeable, but the is esterified with MeOH and the other with
inner one is selectively permeable and regu- the hydrophobic hydrocarbon phytol.
lates the influx and efflux of many metabolites. Chlorophyll b differs from chlorophyll a only
Between the membranes is an intermembrane in having a -CHO group in ring II where
space. Inside the chloroplast is the stroma, a chlorophyll a has a CH3 (Figure 17.2)
protein-rich, gel-like material which contains Because of the long series of conjugated
the enzymes responsible for carbon dioxide double bonds (i.e. alternating double and sin-
fixation and sugar synthesis. Running through gle bonds) in the rings, chlorophylls absorb
the stroma a series of membranes called thy- visible light very strongly indeed, their
lakoids can be seen, and in some regions molar extinction coefficients being amongst
rounded tongues of thylakoid membranes are the highest of any organic compounds.
stacked on top of one another to form grana. Chlorophyll a and chlorophyll b absorb blue
Parts of the grana thylakoids, which are in and red light particularly strongly. However
close contact with one another but separated their absorption spectra do differ slightly from
from the stroma, are known as the appressed one another so that they complement each
Light reactions 219
Outer membrane
Inner membrane
Thylakoids
.------1H-.- Grana
Stroma
Appressed
region
Non-appressed
region
Figure 17.1 Structure of a chloroplast. Thylakoid membranes run through the stroma. In some regions
tongues of thylakoid membranes are stacked on top of one another to form grana. The thylakoid mem-
branes enclose a continuous cavity called the thylakoid space which connects the interiors of the grana to
one another.
other and allow a wider range of wavelengths more light. The carotenoids are also important
of light to be absorbed (Figure 17.3). In higher antioxidants, preventing photo-oxidation of
plants, the average chlorophyll alchlorophyll b chlorophyll at high light intensity
ratio is 3; it is higher than this for PS I and In solution, chlorophyll a absorbs maximal-
lower for PS II. Chlorophyll b is replaced by ly in the red region of the spectrum at 676 nm
chlorophyll c in brown algae and by chloro- and chlorophyll b at 642 nm. However, within
phyll d in red algae. Photosynthetic bacteria the thylakoid membranes chlorophyll mole-
do not contain chlorophyll a but have bacterio- cules are associated with membrane proteins,
chlorophylls a or b. and the wavelength of the absorption maxima
of individual molecules may shift because of
Carotenoids this. In vivo, therefore, individual chlorophyll a
The carotenoids are polyisoprenoid com- molecules may have absorption maxima
pounds with long series of conjugated double between about 660 and 700 nm as a result of
their different environments.
bonds (Figure 17.2b).
There are two classes of carotenoids:
17.3.2 LIGHT ABSORPTION
• carotenes such as ~-carotene which contain
no oxygen; When a molecule absorbs a photon of light, an
• xanthophylls such as lutein which contain electron becomes excited to a higher energy
oxygen. level. The molecule in its excited state may lose
this extra energy in one of several ways:
Carotenoids absorb light of wavelengths
between about 400 and 500 nm and therefore • emitting a photon of lower energy (longer
have absorption spectra which are comple- wavelength) than that absorbed, by the
mentary to the chlorophylls and help to trap process of fluorescence;
220 Photosynthesis
-CH3 in chlorophyll 8
-CHO in chlorophyll b
a Rll
C=O
I
OCH 3
Phytol C20H39
Figure 17.2 Structure of photosynthetic pigments: (a) chlorophyll (chlorophylls a and b differ only in the
nature of Rj);(b) f)-carotene. Both types of pigment contain long systems of conjugated double bonds
which enable them to absorb visible light.
• colliding with a solvent molecule; occur. Thus the absorbed light energy is con-
• transferring energy to another molecule by verted into heat and the solution becomes
resonance energy transfer (RET); warmer. When pigment molecules are embed-
• losing an electron in a chemical reaction. ded in membranes, as in chloroplasts, they are
protected from collision with solvent and may
Excited molecules in solution usually collide be close and at a specific orientation with
with, and pass on their energy to, solvent (often respect to other molecules. These factors favour
water) molecules before any other process can energy transfer by the process of RET.
Light reactions 221
70 -----------_.
60 chlorophyll a
50 \ chlorophy II b
Q)
0
r::::
III
€
5l 30
40
i
f
\/ \
\ ~arotene
~ ! i
/
i
20
i
I
10 !
0
350 450 550 650 750
Wavelength (nm)
Figure 17.3 Absorption spectra of photosynthetic pigments. Both chlorophyll a and b absorb blue light
(wavelength 400 to 470 nm) and red light (600 to 670 nm) but the spectra differ slightly. Carotenoids
absorb mainly between 400 and 500 nm.
17.3.3 RESONANCE ENERGY TRANSFER acts as an energy sink or reaction centre for the
A molecule (the donor) may transfer its excita- complex. This molecule cannot lose energy by
RET and instead loses an energetic electron
tion energy to another nearby molecule (the
which is transferred to another molecule
acceptor) provided that the acceptor absorbs
termed the primary electron acceptor.
light of longer wavelength (lower energy)
than the donor. The migration of energy
17.3.4 THE ELECTRON TRANSPORT SYSTEM
between pigment molecules in the chloroplast
membranes takes place by this process. Numerous studies have shown that photosyn-
The majority of chlorophyll molecules are thesis involves the co-operation of PS I and PS
bound to proteins and form part of light- II. Electrons are transported between these via
harvesting or antenna complexes. These com- an electron transport chain, and the energy of
plexes contain chlorophyll, together with acces- the electrons is increased as a result of light
sory pigments such as carotenoids. The pig- energy absorbed by each photosystem.
ment molecules have different environments The manner in which the two photosys-
and therefore different absorption maxima as terns interact with one another is represented
a result of their interaction with proteins of the by the Z-scheme, which is shown in Figure
complexes. Light absorbed by a molecule in 17.4. In this diagram the vertical axis repre-
the complex can migrate to nearby molecules sents the redox potential, values towards the
which absorb at longer wavelength, and the top are increasingly negative and those
process can continue until the molecule which towards the bottom increasing positive. The
absorbs at the longest wavelength becomes redox potential indicates an electron's energy
excited. In this way all the energy absorbed and how easily it can be transferred to anoth-
anywhere in the complex will eventually er compound. As the addition of an electron is
reach this special molecule which therefore a reduction reaction, compounds high on the
222 Photosynthesis
scheme are strong reducing agents and can In a physical sense the electron transport
transfer electrons to those lower down, reduc- chain consists of several structural units,
ing them in the process. which include the two photo systems and the
Water molecules are split by PS II. This cytochrome b-f complex, which form part of
releases electrons which are excited by the the chloroplast membranes.
light absorbed by PS II and passed via a system
of electron carriers to PS I. The electron trans-
17.3.5 PHOTOSYSTEM I
port process is coupled to the synthesis of ATP
from ADP and Pi' Electrons reaching PS I The units of PS I are found only in the stroma
receive further energy, absorbed by this pho- thylakoids and non-appressed regions of the
to system, and pass into another part of the grana that face the stroma (Figure 17.1). PS I is
electron transport chain where they eventual- a trans-membrane complex consisting of at
ly reduce NADP+ to NADPH. least 13 polypeptides. It consists of a core com-
Primary
acceptor
Ferredoxin-
Primary It.: Fd
NADP+
acceptor Fd
?\U'ta~
Pq NADP+ NADPH
Cytochrome
b-f complex
ADP + Pi
P 700
Photosystem I +-- ~
Figure 17.4 Photosynthetic Z-scheme. Electrons derived from the splitting of water pass through photo-
system II, through the cytochrome b-f complex and through photosystem I before being used to reduce
NADP+. Absorption of light by each photosystem increases the energy of the electrons. ATP is produced
during the passage of electrons between the two photo systems. Electrons from photo system I can be
recycled via ferredoxin and the cytochrome b-f complex in cyclic photophosphorylation.
Light reactions 223
plex and a light-harvesting complex (LHC I), non-appressed regions of the grana and the
which absorbs and channels energy towards stroma thylakoids. PS II consists of a complex
the reaction centre (Figure 17.5). The reaction of about 10 polypeptides which span the
centre of PS I is a chlorophyll a molecule (or membranes in which they are embedded. It is
possibly a pair of chlorophyll a molecules) in a made up of light-harvesting complexes (LHC
special environment and is called P700. II), a reaction centre and a water-splitting unit
(Figure 17.5). The reaction centre is a chloro-
phyll a molecule (or possibly a pair of chloro-
The core complex
phyll a molecules) in a special environment
The main components of the core complex are and is called P680.
two nearly identical proteins: psaA (83 kOa)
and psaB (82 kOa) proteins. The P700 reaction
LHcn
centre, a quinone, an Fe-S complex and some
chlorophyll a and ~-carotene molecules also This consists of a polypeptide (approximately
form part of the core complex. The primary 26 kOa), about six or seven chlorophyll a mol-
acceptor from P700 is called ~ and is the most ecules, about the same number of chlorophyll
powerful known biological reducing agent. It is b molecules and a few carotenoids.
thought to be a special chlorophyll a molecule.
From here the electron is transferred to a
Reaction centre and water splitting
quinone, then to an Fe-S centre and via other
carriers to ferredoxin on the stroma side of the The core of PS II consists of about six polypep-
membrane. Ferredoxin is a red protein of low tides. These include:
molecular weight (approximately 11.6 kOa). It
• 01 and 02 (each approximately 32 kOa) to
is a non-haem iron protein in which the Fe is
which quinones and manganese ions bind
associated with sulphur. Spinach ferredoxin
and which bind the P680 reaction centre itself;
has two Fe atoms bound to two S atoms. One of
• 33-, 17- and 23-kOa proteins which are
the Fe atoms undergoes FeIlL..Fell changes - in
involved in splitting of water;
the reduced state it is a very powerful reducing
• 43- and 47-kOa proteins containing chloro-
agent, able to reduce NADP+ under the influ-
phyll, which also serve as antenna mole-
ence of ferredoxin-NAOP+ reductase, a flavo-
cules.
protein. During this last reaction protons are
consumed on the stroma side of the membrane. Each P 680 molecule receives the energy
absorbed by about 250 chlorophyll molecules,
consisting of roughly equal numbers of chloro-
LHCI
phyll a and chlorophyll b. The primary accep-
This light-harvesting antenna complex sur- tor of PS II is thought to be pheophytin, a
rounds the core and contains about 80 mole- chlorophyll molecule lacking a magnesium
cules of chlorophyll a and 20 of chlorophyll b. atom at its centre.
These absorb light and, through RET, direct the PS II is also responsible for the water-split-
energy towards the pigment molecules of the ting reaction which can be summarized as fol-
core complex and hence to the reaction centre. lows:
2H20--->02 + 4H + + 4e-
17.3.6 PHOTOSYSTEM II
This process is very poorly understood but
The units of PS II are concentrated in the both Mnll and Cl- are required. Synthetic elec-
appressed regions of the grana thylakoids, tron donors such as semicarbazide and hydrox-
with only small amounts being found in the ylamine can substitute for water in this reaction.
Stroma
H+
A'tP
NADPH + H+ syn~$e
~ATP
NADP+ + 2H+
2H+
Cytoc;hJ;0m9b-f
PS II compleX ~ADP+
Pi
Ferredoxin
CFo Thy/akoid
membranlll
Water-splitting - 2H+
complex 2e
H+
H20 L 11.02 + 2H+ Plastocyanin Thylakoid space
Figure 17.5 Diagrammatic representation of the complexes involved in photosynthesis. Passage of electrons along the electron transport
chain moves protons from the stroma into the thylakoid space and thus creates a proton gradient. This is used to drive the synthesis of ATP.
Plastoquinone, plastocyanin and ferredoxin are mobile and can transport electrons between the complexes.
ATP production 225
The part of the unit responsible for the reac- reduce NAOP+ leaves PS I short of an electron
tion contains four manganese atoms which (i.e. oxidized) and it is dependent on PS II to
undergo a cycle of oxidation changes as elec- replace it. Until this has happened no further
trons are extracted from water and passed on electrons can be lost. Similarly, the electron
to the P68o molecule, which has lost its electron. lost from PS II must be replaced by electrons
The 33-, 17- and 23-kOa proteins and a tyro- released during the splitting of water. Thus,
sine residue on the 01 protein appear to be the chain behaves as though electrons flow
involved in this process. through it from water to NAOP+.
The primary acceptor for PS II, to which an The locations of the PS I, PS II and ATP syn-
electron is transferred from P680 , is pheo- thase complexes are relatively fixed: they are
phytin. From here the electron is transferred located in different regions of the thylakoid
to a molecule of plastoquinone (Pq) which is membranes. However, some of the electron
permanently bound to 02 and which carriers are mobile and are able to transfer
becomes partially reduced. The electron from electrons between the fixed complexes. Thus
this molecule is passed to another plasto- reduced plastoquinone, plastocyanin and
quinone molecule bound to 01. This molecule ferredoxin can all move. The cytochrome b-f
remains bound until it has accepted a second complex may also be mobile and move
electron to fully reduce it, after which it is between stacked and unstacked regions of the
released. By this means the two electrons thylakoids.
needed for the reduction of plastoquinone
can be supplied, one at a time, by pheophytin.
17.5 ATP PRODUCTION
17.3.7 CYTOCHROME b-f COMPLEX The components of the electron transport

chains are embedded in the thylakoid mem-
This consists of a complex of four subunits: branes in such a way that reactions in which
cytochrome f (33 kOa), cytochrome bS63 (23 H+ are used up take place on the stroma side
kOa, containing two haems, also called of the membrane, and those in which H+ are
cytochrome b6), Fe-S protein (20 kOa) and released take place on the thylakoid space
another 17-kOa chain. The complex spans the side. This results in development of a gradient
membrane and pumps protons across it as of [H+] across the membrane, as shown in
electrons flow from plastoquinone to plasto- Figure 17.5. The pH of the thylakoid space
cyanin. Cytochrome b allows two electrons to may fall to around pH 4.
be accepted from plastoquinone and handed In addition to the electron transporting
on one at a time to plastocyanin. complexes described above, an additional
complex, ATP synthase, is also embedded in
17.3.8 PLASTOCYANIN the thylakoid membranes. This resembles the
corresponding enzyme present in mitochon-
This is a blue ll-kOa copper-containing pro-
dria and consists of CFo and CF] components.
tein. The copper atom co-ordinates with the S
CF0spans the membrane and CF] protrudes on
atoms of a methionine and cysteine residue
the stromal side, and is the part which actual-
and with two histidine residues.
ly synthesizes ATP from AOP and Pi' The pro-
ton gradient, generated during electron trans-
17.4 INTEGRATION OF THE ELECTRON
port, is used to drive the synthesis of ATP from
TRANSPORT SYSTEM
AOP and Pi. These processes are entirely anal-
Operation of the electron transport chain ogous to those which result in ATP production
requires PS I and PS II to work together. during electron transport in mitochondria
Removal of an excited electron from PS I to (Chapter 12).
226 Photosynthesis
Although it is known that the flow of elec- tion. The reaction is catalysed by the enzyme
trons between PS II and PS I generates the pro- RUBP carboxylase/oxygenase, which is usually
ton gradient needed to produce ATP, it is not known as Rubisco. Rubisco catalyses the reac-
easy to determine the number of ATP mole- tion of ribulose-1,5-bisphosphate with carbon
cules produced for each pair of electrons trans- dioxide to form, initially, an unstable, six-car-
ported. This is because two processes can give bon compound, which rapidly breaks down to
rise to ATP: form two molecules of 3-phosphoglyceric acid
(Figure 17.7). Rubisco is located on the stromal
• non-cyclic photophosphorylation;
surface of the thylakoid membranes. It is the
• cyclic photophosphorylation.
commonest protein on earth, making up about
The non-cyclic process is that already 50% of the soluble protein in leaves. A hectare
described, in which electrons are transported of lawn contains between 5 and 7 kg of pure
from water by PS II to PS I and used to reduce enzyme. The reaction which it catalyses
NADP+. If insufficient NADP+ is available to requires the presence of magnesium. The
accept electrons from PS I then they may be enzyme is not totally specific for carbon diox-
diverted into a cyclic process during which ide and will also catalyse a reaction with 02
they are passed back to the cytochrome b-f instead of CO 2• This reduces photosynthetic
complex and hence back to PS I. This results in efficiency, as discussed later in this chapter.
production of ATP but no NADPH is formed. The next two steps use ATP and NADPH
This process is represented by the dotted line and depend on the light reactions for produc-
in Figure 17.4. tion of these compounds. These reactions pro-
The dark reactions of photosynthesis duce glyceraldehyde-3-phosphate. Some of
require three molecules of ATP and two mole- this is used to regenerate ribulose-1,5-bisphos-
cules of NADPH for each molecule of carbon phate by a rather complex series of reactions
dioxide fixed. Cyclic photophosphorylation so that the cycle can continue. These reactions
appears to operate to a variable extent, and are catalysed by the enzymes transaldolase
this may allow the proportion of ATP and and transketolase, and the steps are virtually
NADPH produced to be adjusted to meet the identical to parts of the pentose phosphate
demands of the dark reactions. pathway. Six molecules of ribulose-l,5-bispho-
sphate (6 x 5 carbon atoms) and six molecules
of carbon dioxide (6 x 1 carbon atoms) give
17.6 THE DARK REACTIONS (CALVIN CYCLE)
rise to 12 molecules of glyceraldehyde-3-phos-
Operation of the light reactions results in the phate (12 x 3 carbon atoms). Ten molecules of
release of NADPH and ATP into the chloro- glyceraldehyde-3-phosphate are used to re-
plast stroma. These compounds are used to form six molecules of ribulose-l,5-bisphos-
convert carbon dioxide into sugars, in a phate, and two molecules are left over. These
process which does not itself depend on light. represent the net output of the cycle.
The reactions involved make up the Calvin The sugars produced by the Calvin cycle
cycle, named after its discoverer Melvin are either converted into starch within the
Calvin. This cycle is shown in Figure 17.6. chloroplasts, or exported as triose phosphate
Reactions of the Calvin cycle are catalysed (glyceraldehyde-3-phosphate) into the cyto-
by enzymes in solution in the stroma or on the plasm. Starch accumulates during periods of
stromal side of the thylakoid membranes. The illumination but may be broken down and the
crucial first step, in which carbon dioxide carbon exported into the cytoplasm during
reacts with ribulose-1,5-bisphosphate (RUBP), darkness. The starch therefore acts as a tempo-
converts inorganic carbon into an organic rary store of carbon during times of rapid car-
form and is hence referred to as carbon fixa- bon fixation.
The dark reactions (Calvin cycle) 227
t
Glyceraldehye-
3-phosphate
(12 x C3)
CO2 12NADP+
[3)
12NADPH + 12H+
1,3-diphospho- Dihydroxy-
t
glycerate acetone
(12 x C 3) phosphate
(3 x C 3) PRODUCT
12ADP
[2]
12ATP Fructose-1,6-
bisphosphate
3-phospho- (3 x C6)
glycerate
(12 x C3 ) [6] ~
~~~
Fructose-6-
phosphate
[1,
(2 x C6)
Ribulose-1,5-
bisphosphate
(6 x Cs) Erythrose-4-
phosphate
(2 xC4)
~
6ADP
[13) [8)
6ATP
Sedoheptulose-
Ribulose-S- 1,7-bisphosphate
phosphate (2 x C7)
(6 x Cs)
~
Xylulose-S-
phosphate
(2 x Cs)
Xylulose-5- Sedoheptulose-7-
phosphate [10) phosphate
(2 x Cs)
~ (2xC7)
Ribose-S- Glyceraldehye-3-
phosphate phosphate
(2 x Cs) (2 x C,)
Figure 17.6 The Calvin cycle. Reaction of ribulose-I,5-bisphosphate with carbon dioxide yields 3-phos-
phoglycerate which is phosphorylated and reduced to form glyceraldehyde-3-phosphate. Most of this is
used to regenerate ribulose-I,5-bisphosphate by reactions which are very similar to those of the pentose
phosphate pathway, but the remainder is converted to sugars. Enzymes catalysing the reactions are as
follows: [1] ribulose I,5-bisphosphate carboxylase/oxygenase (Rubisco), [2] phosphoglycerate kinase, [3]
glyceraldehyde-3-phosphate dehydrogenase, [4] triose phosphate isomerase, [5] fructose bisphosphate
aldolase, [6] fructose bisphosphatase, [7] transketolase, [8] aldolase, [9] sedoheptulose bisphosphatase,
[10] transketolase, [11] ribulose phosphate 3-epimerase, [12] ribulose phosphate isomerase, [13] phospho-
ribulokinase.
228 Photosynthesis
CH 2 0®
I
H-C-OH
I
C0 2 H
3-phosphoglycerate
CH 2 0®
I 3-phosphoglycerate
C=O
I
H-C-OH
I
H-C-OH
I
CH 2 o®
CH 2 o®
Ribulose-1,5- I
C0 2 H
bisphosphate
phosphoglycolate
3-phosphoglycerate
Figure 17.7 The reactions catalysed by Rubisco. This enzyme can catalyse reaction of ribulose-l,5-bisphos-
phate with either carbon dioxide or oxygen. The former reaction produces two molecules of 3-phospho-
glycerate as part of the Calvin cycle. The latter reaction produces one molecule of 3-phosphoglycerate
and one of phosphoglycolate which is used in photorespiration.
The inner membrane of the chloroplast is Calvin cycle and it must enter the chloroplasts
practically impermeable to polar molecules as triose phosphates move into the cytoplasm.
such as free sugars, Pi and triose phosphates, When Pi is not available from the cytoplasm,
and specific carriers are needed to transport starch accumulation in the chloroplasts is
some of them across the membrane. A carrier favoured. This process liberates Pi which can
which facilitates the counter-transport of be re-used in the Calvin cycle.
triose phosphates and Pi appears to be partic- Some of the glyceraldehyde-3-phosphate
ularly important in regulating the movement may be converted to dihydroxyacetone phos-
of the products of photosynthesis out of the phate. These two molecules then condense to
chloroplast into the cytoplasm. Pi is required form fructose-l,6,-bisphosphate, from which
for the synthesis of organic molecules by the fructose-6-phosphate, glucose, starch and
Photorespiration 229
other sugars may arise by reactions described ferredoxin converts thioredoxin into its
in Chapter 18. reduced (-SH) form. This activates a number of
Operation of the pathway may be consid- Calvin cycle enzymes, including glyceralde-
ered to bring about conversion of six molecules hyde-3-phosphate dehydrogenase, fructose
of carbon dioxide to one molecule of fructose- bisphosphatase, sedoheptulose bisphos-
6-phosphate. The overall equation for opera- phatase and phosphoribulokinase. It may also
tion of the Calvin cycle can be obtained by inhibit enzymes involved in sugar breakdown.
adding together the steps shown in the cycle The rate of conversion of triose phosphates
(Figure 17.6). The equation which results is: into sucrose in the cytoplasm is also regulated
by fructose 2,6-bisphosphate, which has a sim-
6C02 + 12NADPH + 12H+ + 18ATP +
ilar role in control of glycolysis and gluconeo-
11HzO -> fructose-6-phosphate + 12NADP+
genesis in animals. This regulation appears to
+ 18ADP + 17Pj be complex, but in general the compound
accelerates conversion of fructose-6-phos-
phate into fructose-1,6-bisphosphate and
17.7 CONTROL OF PHOTOSYNTHESIS
inhibits the reverse reaction catalysed by fruc-
Many factors influence the rate of photosyn- tose-1,6-bisphosphatase. By this mechanism
thesis. Light is the most important of these, the relative rates of sugar synthesis and break-
and some of its effects are listed below: down are changed.
• a direct effect on the light reactions, leading
to increased availability of ATP, NADPH, 17.8 PHOTORESPIRATION
etc.;
Plant cells contain mitochondria and therefore
• a long-term effect on the development of
respire to produce ATP. In the process they
chloroplasts and the induction of photosyn-
consume oxygen and produce carbon dioxide.
thetic enzymes;
This occurs in both green and non-green tis-
• effects on the activity of Calvin cycle and
sues and is not dependent on light (except
other enzymes.
indirectly, in that photosynthesis provides
At least five Calvin cycle enzymes (Rubisco, sugars which are substrates for respiration).
glyceraldehyde-3-phosphate dehydrogenase, However, in many plants another type of res-
fructose bisphosphatase, sedoheptulose bis- piration, photorespiration, also takes place.
phosphatase and phosphoribulokinase) are Photorespiration is dependent on light and is
activated by light. Several factors may con- often much faster than mitochondrial respira-
tribute to this activation; for example, changes tion. Photorespiration involves the production
in pH resulting from proton movement from and metabolism of glycolate by a process
the stroma into the lumen of the chloroplasts which is sometimes called the glycolate cycle.
may activate enzymes such as Rubisco. In addi- Photorespiration is closely linked to the Calvin
tion, compounds such as NADPH and ATP cycle, as the enzyme Rubisco is common to
formed in the light reactions are positive both pathways.
allosteric effectors of Rubisco and of glycer- Rubisco can catalyse the reaction of either
aldehyde-3-phosphate dehydrogenase. A fur- carbon dioxide or oxygen with ribulose l,5-bis-
ther very important effect is brought about phosphate. Reaction with carbon dioxide (car-
through the action of light effect mediators. boxylase activity) produces two molecules of
These are proteins containing cysteine residues 3-phosphoglycerate, but reaction with oxygen
that can exist either in the -SH form or in the (oxygenase activity) produces 3-phosphoglyc-
oxidized -S-S- form. The best known of these erate and phospho glycolate (Figure 17.7). The
is called thioredoxin. In the light, reduced former can be used for the synthesis of sugars
230 Photosynthesis
but the latter undergoes oxidation in which and molecular oxygen, has a very high affinity
carbon dioxide is produced. This oxidation for oxygen and is completely saturated even at
involves the chloroplasts, peroxisomes and very low oxygen concentrations. Thus at low-
mitochondria, as shown in Figure 17.8. ered oxygen concentrations, rates of photores-
During photorespiration oxygen is con- piration can be reduced without affecting
sumed and carbon dioxide is produced. Some mitochondrial respiration. Young plants
of the ribulose l,5-bisphosphate which could grown in 2-5% 0z instead of the normal 20%
have been used in the Calvin cycle to produce therefore grow about twice as fast as normal.
sugars is converted back to carbon dioxide. Carbon dioxide enrichment of the atmosphere
Photorespiration therefore acts in the opposite is commonly used in growing protected crops.
sense to photosynthesis, and the operation of It not only increases rates of photosynthesis,
this pathway reduces the net efficiency of con- but decreases rates of photo respiration by
version of carbon dioxide into sugars. Under competing with oxygen for Rubisco.
many circumstances photorespiration occurs The relative importance of photo respiration
at 20-25% of the rate of photosynthesis, and it increases with increasing light intensity, tem-
is a major factor which limits the photosyn- perature and pH. As temperature rises the sol-
thetic efficiency of many plants. ubility of carbon dioxide in water decreases
Carbon dioxide and oxygen compete for faster than that of oxygen. Increasing pH
reaction at the active site of Rubisco. High lev- decreases the availability of carbon dioxide as
els of carbon dioxide favour the Calvin cycle, it is converted to HC0 3- at high pH. In bright
whilst high levels of oxygen favour photores- light the pH of the stroma is increased and this
piration. In the atmosphere the ratio of COz:Oz increases the rate of photorespiration, as
is very low (0.02:20%) so it may be inevitable described above. Photorespiration is thus a
that some ribulose bisphosphate oxygenase particular problem for plants growing in
activity will occur. Photorespiration may, bright light and at high temperatures, and
under these circumstances, be an attempt by many plants show specialized adaptations to
the cell to convert some phosphoglycolate growth under these circumstances.
back into sugars. The fact that plants grow faster when pho-
In photorespiration, one molecule of NHt is torespiration is reduced, and that some of the
produced from glycine for each molecule of most productive plants do not carry out the
COz released. This NHt is rapidly reassimilat- process at all, suggests that it may be possible to
ed into organic compounds by the reactions increase the efficiency of crop growth by
described in Chapter 20, and this constitutes decreasing rates of photorespiration. This
the photorespiratory nitrogen cycle. Much of might be achieved through using chemical
the ammonia converted into organic com- inhibitors of glycolate oxidase such as a-
pounds originates in this way. hydroxypyridine methanesulphonate or 2-
hydroxy-3-butynoic acid. It might also be
achieved by changing the structure, and there-
17.8.1 FACTORS AFFECTING RATES OF
fore the specificity, of the active site of Rubisco
PHOTORESPlRATION
by conventional breeding or by genetic engi-
Reducing the oxygen content of the air neering. By specific modification of individual
reduces the rate of photorespiration because bases in the genes which code for Rubisco it is
Rubisco has a low affinity for oxygen and is possible to change the amino acids at the active
not saturated even at concentrations of oxy- site of the enzyme (see Chapter 21), and this
gen found in the air. In mitochondrial respira- may alter its specificity so that it reacts less read-
tion, however, cytochrome oxidase, which ily with oxygen. However this is difficult to
catalyses the reaction between cytochromes achieve because of the complexity of Rubisco,
Photorespiration 231
Chloroplast
CH 2c<E)
6=0
H-6-0H
H-6-0H
tH2o® glycolate
3-phosphoglycerate
glycolate
glyceric acid glutamate~
i a.-ketoglutarate-1
Peroxisome
CH 20H
CH 2NH 2
6H 2NH 2
602 H
602 H
serine glycine
CH 20H x2 CH 2NH 2
6H 2NH 2
60 2H
( 602 H Mitochondrion
NH3 glycine
serine CO2
Figure 17.8 The pathway of photorespiration. Phosphoglycolate is produced by reaction of ribulose-l,5-

bisphosphate with oxygen and is then oxidized, with production of carbon dioxide, in a series of reac-
tions which involve the chloroplast, peroxisome and mitochondrion. One effect of this process is to
degrade ribulose-l,5-bisphosphate which would otherwise be used in the Calvin cycle, and thus to
reduce net photosynthetic sugar production.
which has a molecular weight of about 560 kDa. kDa each, and eight small subunits, each about
It consists of eight large subunits, of about 56 14 kDa. The sequence of amino acids in the
232 Photosynthesis
large subunits is encoded in the chloroplastic malate by the action of malate dehydrogenase.
DNA and the small subunits in nuclear DNA. The malate moves to the bundle-sheath cells,
The latter proteins are therefore synthesized in where it is acted on by malic enzyme to pro-
the cytoplasm and must be imported into the duce pyruvate and CO 2 , NADP+ is reduced to
chloroplasts where they form a complex with NADPH. The carbon dioxide released reacts
large subunits to form active enzyme. Not only with ribulose l,S-bisphosphate in the Calvin
must the genes be modified in the correct way, cycle using the enzyme Rubisco. The pyruvate
but they must also direct their products to the passes back to the mesophyll cells and is con-
correct location in the cell. verted to phosphoenolpyruvate at the expense
of two high-energy phosphate bonds in a reac-
tion catalysed by pyruvate phosphate dikinase.
17.9 PHOTOSYNTHESIS IN C4 PLANTS
This system acts as a 'pump' for carbon
The major crop plants of temperate regions of dioxide. In contrast to Rubisco, phospho-
the world fix carbon dioxide using the Calvin enolpyruvate carboxylase has a very high
cycle. Photorespiration occurs in these plants affinity for carbon dioxide and none for oxy-
so that a significant proportion of their poten- gen. The uptake of carbon dioxide is thus
tial yield is lost. rapid and specific. When the carbon dioxide is
In other plants, typically tropical grasses delivered to the Rubisco, oxygen is excluded,
which grow under high light intensities and at eliminating formation of phosphoglycolate
high temperatures, a modified type of photo- and photorespiration.
synthesis takes place and photorespiration is In the Calvin cycle each molecule of carbon
virtually undetectable, so yields of organic dioxide fixed requires two NADPH and three
matter are much higher. ATP molecules. In the C4 mechanism a further
These tropical plants use the Hatch and two high-energy phosphate bonds are broken
Slack or C4 pathway for photosynthesis. Here in forming phosphoenolpyruvate (ATP is con-
the initial products of carbon dioxide fixation verted to AMP). In plants growing at high
are four-carbon compounds, hence the name light intensities ATP is readily available from
C4 pathway. In contrast, plants using the the light reactions, so the extra ATP require-
Calvin cycle are called C3 plants as three-car- ment of the C4 pathway may be less important
bon compounds (3-phosphoglycerate and than losses resulting from photorespiration. In
glyceraldehyde-3-phosphate) are the initial many tropical crops, therefore, C4 photosyn-
products of carbon dioxide fixation. thesis is favoured. In temperate climates light
The C4 pathway involves the cooperation of is more likely to be a limiting factor so it may
two types of cell - mesophyll cells and bundle be more efficient for plants to economise on
sheath cells. The mesophyll cells occur towards use of ATP, and under these conditions the C3
the outside of the leaf and are freely accessible pathway may be preferred.
to the air. In these cells carbon dioxide is initial- Another advantage to the plant results from
ly fixed into C4 compounds, which are then use of the C4 route. As phosphoenolpyruvate
transported into the bundle sheath before carboxylase has a very high affinity for carbon
being broken down to re-release carbon dioxide dioxide it is able to obtain sufficient carbon
which enters the Calvin cycle (Figure 17.9). dioxide even when the stomata are very near-
The chloroplasts of the mesophyll cells con- ly closed. Under arid conditions this allows the
tain the enzyme phosphoenolpyruvate car- plants to conserve water more effectively than
boxylase which catalyses the reaction between if they were using the C3 pathway.
phosphoenolpyruvate and carbon dioxide. The efficiency of the C4 process is seen
The oxaloacetate produced is then reduced by when the rates of photosynthesis of different
NADPH (produced in the light reactions) to plants are compared (Table 17.1).
Crassulacean acid metabolism 233
Mesophyfl cell
phosphoenol oxaloacetate
AMP~JPyruvate ~NADPH+H+
ATP-/ ~NADP+
pyruvate malate
pyruvate malate
NADPH + H+~--~ CO2

NADP+
Bundle sheath cell
Figure 17.9 Photosynthesis in C4 plants. This involves initial reaction of carbon dioxide with phosphoenol
pyruvate in the mesophyll cells in a reaction catalysed by phosphoenolpyruvate carboxylase. The four-
carbon compound, malate, moves to the bundle sheath cells where it is degraded to release carbon diox-
ide which enters the Calvin cycle. The process requires more ATP than C3 photosynthesis but, because of
the high specificity of phosphoenolpyruvate carboxylase, prevents photorespiration from taking place.
17.10 CRASSULACEAN ACID METABOLISM known for a long time to increase their acid
content at night and to decrease it in the day.
This type of photosynthesis is common Normally the stomata are open during the
amongst succulent plants and some bromeli- night but closed in the day. At night, carbon
ads, lilies and orchids. These plants have been dioxide is fixed in the chloroplast-containing
234 Photosynthesis
Table 17.1 Typical rates of carbon dioxide fixation 17.11 HERBICIDES AND PHOTOSYNTHESIS
in different types of plant
Because photosynthesis is of vital importance
Type RateofCOz to plants but does not occur in animals it is a
fixation very obvious target for the development of
(mgCO z potential herbicides, and a number of impor-
dm-z h-1) tant herbicides function by inhibiting the
process.
Slow-growing desert species, orchids etc 1-10
Tropical and subtropical evergreens, 5-15 The dipyridinium herbicides such as
temperate evergreens diquat and paraquat act on PS I. They divert
Most temperate herbs and deciduous 15-30 electrons away from NADP+ in the region of
trees ferredoxin, and the herbicide molecules
Rapidly growing temperate agronomic 20-40 themselves become reduced in the process.
crops (wheat, soyabean, etc.) As the reduced herbicide is reoxidized, super-
C4 tropical grasses (maize, sugarcane etc.) 50-90 oxide radicals are generated and these are
very toxic, causing extensive damage by
cells of the photosynthetic leaf or stem tissue, reacting with a wide range of molecules pre-
and converted to oxaloacetate through the sent in the cell.
action of phosphoenolpyruvate carboxylase. The D1 protein of PS II is thought to be the
Much of this is converted to L-malic acid site of action of substituted urea herbicides
which is stored in the large vacuoles that are such as monuron (now superseded) and
characteristic of these plants. During the day diuron, which thus prevent plastoquinone
malic acid is decarboxylated to form pyruvate reduction. This is of considerable interest to
or phosphoenolpyruvate under the action of genetic engineers, as alteration of the struc-
one of several enzymes, which differ from ture of this protein may make crop plants
species to species. The carbon dioxide pro- resistant to these herbicides. Weeds contain-
duced enters the Calvin cycle, where it is used ing the normal protein would remain suscep-
to produce starch and sucrose in the normal tible to the herbicides. Such modified D1 pro-
way (Figure 17.10). teins may be in found herbicide-resistant
Pineapple is the main agricultural crop plants or may be produced by deliberate
using this type of photosynthesis. This mecha- modification of the gene coding for the pro-
nism allows the stomata to remain closed dur- tein. Introduction of such modified genes
ing the day, which conserves water. This into crops would allow the use of potent,
achieves the same purpose as C4 photosynthe- wide-spectrum herbicides to control weeds in
sis, but in this case the Calvin cycle and initial these crops.
carbon dioxide fixation are separated in time
rather than space.
Herbicides and photosynthesis 235
CO2
Phosphoenol
pyruvate
~ Oxaloacetate
phosphoenolpyruvate
carboxylase
Vacuole
phosphoenolpyruvate
carboxykinase
T
Phosphoenolpyruvate Oxaloacetate ~ Malate
Malate dehydrogenase
daytime
Figure 17.10 Crassulacean acid metabolism. In some plants, carbon dioxide is converted into four-carbon
acids such as malate during darkness. It is released from these compounds in the light and is converted
into sugars by the Calvin cycle.
GLUCONEOGENESIS AND 18
CARBOHYDRATE SYNTHESIS

18.1.1 Starting materials 237
18.1.2 Outline of the pathway 238
18.1.3 Differences between gluconeogenesis 238
and glycolysis
18.2 Gluconeogenesis via pyruvate 238
18.2.1 The control of pyruvate production 238
and use
18.3 The production of 240
fructose-1,6-bisphosphatase
18.4 The hydrolysis of 240
fructose-1,6-bisphosphate
18.4.1 The control of phosphofructokinase 240
and fructose-1,6-bisphosphate
18.4.2 The fate of fructose-6-phosphate 242
18.5 The utilization of glucose-6-phosphatase 243
18.6 Gluconeogenesis from propionate 243
18.7 The synthesis of complex carbohydrates 243
18.7.1 Disaccharides 243
18.7.2 Synthesis of polysaccharides 246
18.1 INTRODUCTION have a large requirement for glucose as an

energy source in the tissues and, in the case of
Gluconeogenesis is the process which pro-
duces glucose from simpler molecules. In the lactating animal, for making lactose.
plants it is one of the principal routes leading
18.1.1 STARTING MATERIALS
to the eventual synthesis of complex carbohy-
drates. Animals divide into two groups as far Many metabolites can be used as starting
as the importance of gluconeogenesis is con- materials for gluconeogenesis, but some sim-
cerned. Simple-stomached animals can usual- ple compounds cannot. Three-carbon com-
ly rely on a supply of glucose in their food pounds such as pyruvate, phosphoenolpyru-
from the breakdown of a-linked carbohy- vate or triose phosphates are all capable of
drates such as starch. Apart from a small being transformed into glucose and are
amount of glycogen synthesis, their glucose termed glucogenic materials. The reaction in
metabolism is principally one in which it is which acetyl-CoA is produced from pyruvate
oxidized. On the other hand, ruminant ani- (pyruvate dehydrogenase) is irreversible in
mals receive very limited supplies of glucose organisms lacking the glyoxylate cycle. Thus
from the gut and rely on synthesis from the acetyl-CoA and related compounds (fatty
volatile fatty acids, mainly propionate. The acids, ketone bodies, ketogenic amino acids)
pathway is very active because ruminants cannot be used for glucose synthesis.
238 Gluconeogenesis and carbohydrate synthesis
18.1.2 OUTLINE OF THE PATHWAY enzyme pyruvate carboxylase. This reaction
requires the hydrolysis of a molecule of ATP in
In essence the pathway is very similar to a
order to overcome part of the energy barrier to
reversal of glycolysis. It starts with two mole-
the pathway.
cules of a three-carbon compound. After a
In the next step, phosphoenolpyruvate car-
series of transformations, these become one
boxykinase catalyses the decarboxylation of
molecule each of dihydroxyacetone phos-
oxaloacetate to produce phosphoenolpyruvate.
phate and glyceraldehyde-3-phosphate which
This step also requires energy to drive it, in this
are linked to give fructose-1,6-bisphosphate
case in the form of GTP. This means that the
(Figure 18.1). The final part of the pathway is a
conversion of pyruvate to phosphoenolpyru-
rearrangement to yield glucose-6-phosphate
vate uses two high-energy phosphate groups,
(Figure 18.2).
whereas the reverse reaction produces only
one. If both of these sequences of reactions
18.1.3 DIFFERENCES BETWEEN were active at the same time, the overall effect
GLUCONEOGENESIS AND GLYCOLYSIS would be that one mole of ATP and one of GTP
would be used, and only one mole of ATP
There are three steps in glycolysis that are
would be produced. This leads to the potential
essentially irreversible because they are
for a futile cycle, as illustrated in Figure 18.3b.
accompanied by large, negative, free energy
(The term 'futile cycle' is well-accepted in bio-
changes:
chemistry but it may be a misnomer - under
• hexokinase/glucokinase some circumstances these reaction sequences
• phosphofructokinase can have important functions in the overall
• pyruvate kinase. energy metabolism of warm-blooded animals;
see Chapter 29.)
The pathway for glucose synthesis has to
avoid these steps by a series of 'by-pass reac-
tions'. These steps have a further use in pro-
18.2.1 THE CONTROL OF PYRUVATE
viding points at which the flow through the PRODUCTION AND USE
pathway can be controlled.
The most important controls of the steps
illustrated in Figure 18.3 are exerted through
18.2 GLUCONEOGENESIS VIA PYRUVATE
the activities of the allosteric enzymes pyru-
Under normal circumstances pyruvate itself is vate kinase and pyruvate carboxylase. The
not found in high concentrations in cells, but activity of pyruvate kinase is greatly inhibited
other precursors such as malate, lactate and by the presence of high levels of ATP or
glucogenic amino acids must pass through this NADH. On the other hand, pyruvate car-
metabolite. Due to the irreversibility of the boxylase is almost inactive in the absence of
enzyme pyruvate kinase, the direct produc- acetyl-CoA.
tion of phosphoenolpyruvate from pyruvate is
not possible, and a two-stage pathway is used
18.3 THE PRODUCTION OF FRUCTOSE-l,6-
to by-pass the obstruction (Figure 18.3). This
BISPHOSPHATE
short sequence is complicated by the fact that
it takes place in different parts of the cell. The conversion of phosphoenolpyruvate to
Pyruvate, produced in the cytosol, has first to fructose-1,6-bisphosphate is an exact reversal
be taken up by the mitochondrion where it is of the equivalent steps in glycolysis (see Figure
carboxylated using bicarbonate ions to form 18.1). The free energy changes for all of these
oxaloacetate under the influence of the reactions are quite small.
The production of fructose-1,6-bisphosphate 239
O~
C-OH
I
Phosphoenolpyruvate c-o@
II
C
H/ 'H
1}::::!- H20
r ~ Enolase
O~
2-phoaphoglycerate
C-OH
I
HC-O@
I
CHpH
3-phoaphoglycerate
o
~
H
C-OH
Phosphoglycero
mutase
I
HCOH
I
CHP@
ATP
Phosphoglycerate
ADP kinase
1,3-blaphoaphoglycerate O~
C-O@
I
NADH + H+ HCOH
I
CH 20®
'Glyceraldehyde 3·phosphate
dehydrogenase
®
P OCH2
dlhydroxyacetone
phoaphate
HC
I
=0 ')C = 0
HCOH HOCH 2
I
CH 20®
Glyceraldehyde-3-phoaphate
®OCH 2 0 CH20®
~ OH Fructose-1,6-blsphosphate
Figure 18.1 Pathway for the synthesis of fructose-l,6-bisphosphate from phosphoenolpyruvate. The
steps are essentially a reversal of the equivalent ones in glycolysis (3-GPA = 3 phosphoglycerate).
° ADP. Figure 18.4 illustrates the futile cycle that
9
®OCH 2 CH 20®
could result if both of these reactions occurred
at the same time. The overall reaction, shown
F,uctos.-1,6-blsphosph." as the sum of the two reactions, would proceed
OH very readily because the free energy change for
the breakdown of ATP to ADP and phosphate
l Fructose-t,6-bisphosphatase is large and negative.
!'- PI
This pair of reactions provides the most
important control mechanism for the flow of
material between glucose and pyruvate. The
®OCH 2 0 CHpH reactions catalysed by phosphofructokinase
~"~ and by fructose-1,6-bisphosphatase are never
~ Fructoa.-6-phoaph.t.
allowed to take place at the same time. If phos-
OH phofructokinase is operative then glycolysis
will take place; if fructose-1,6-bisphosphatase
is active then the metabolic flow will be
Jr Phosphog/ucose isomerase towards glucose synthesis. Both of the
enzymes are allosteric.
CHP@
k ~"
HO~OH
Glucoa.-6-phoaph.t. 18.4.1 THE CONTROL OF
PHOSPHOFRUCTOKINASE AND FRUCTOSE-
OH 1,6-BISPHOSPHATASE
l G/ucose-6-phosphatase
The step catalysed by phosphofructokinase is
!'-P 1
the first one in which the substrate is
absolutely committed to being broken down
by glycolysis. Prior to this step the phospho-
HO
~::OH~ OH
Glucos.
rylation of glucose and the rearrangement of
glucose-6-phosphate to fructose-6-phosphate
are both reactions that are used for other pur-
OH poses, such as the interchange of sugars or the
pentose phosphate pathway. Inhibiting the
Figure 18.2 Pathway for the conversion of fructose- activity of phosphofructokinase will therefore
1,6-bisphosphate into glucose-6-phosphate. The
steps catalysed by phosphatases overcome the selectively 'switch off' the process of glycoly-
unfavourable energy changes which would be sis. In looking at possible control mechanisms,
needed to reverse the kinase-catalysed reactions of it is clear that the main purpose of glycolysis is
glycolysis. the eventual production of energy in the form
of ATP. If ATP is abundant then there is little
point in the pathway functioning. However, if
18.4 THE HYDROLYSIS OF FRUCTOSE-l,6-
the converse is true and most ATP has been
BISPHOSPHATE
hydrolysed to ADP, or even AMP, then the
In glycolysis, phosphofructokinase uses ATP to cell may be regarded as being deficient in
add the phosphate group to fructose-6-phos- energy. Accumulation of citrate indicates that
phate. In glucogenesis, the reverse reaction there is a low demand for the oxidation of glu-
involves the simple hydrolysis of a phosphate cose by glycolysis. The levels of ATP, ADP,
group by the enzyme fructose-1,6-bisphos- AMP and citrate define the circumstances
phatase. This reaction does not phosphorylate when glycolysis ought to be inactive or active.
The hydrolysis of fructose-1,6-bisphosphate 241
ATP COOH
I
XGDP+Co, C-O@)
II
ADP
CH
GTP Pho.pho.nolpyruvat.
Phosphoenolpyruvate
cerboxyklnase ADP
COOH
I
C=O
I
CH 2
I
COOH ATP
O.aloae.tat.
GLUCOGENESIS GLYCOLYSIS
co2 COOH
I
C=O
ATP I
CH 3
Pyruvate
a. pathways for the

interconversion of pyruvate
and phosphoenolpyruvate.
b. futile cycle based upon

the above pathway.
ATP
Figure 18.3 Pathways for the interconversion of pyruvate and phosphoenolpyruvate. If all of the
enzymes were active at the same time, these reactions would form a futile cycle by hydrolysing two mol-
ecules of ATP but forming only one.
Accordingly, phosphofructokinase activity is Phosphofructokinase is active when ATP and

insignificant when ATP or citrate concentra- citrate levels are low, and AMP and/or ADP
tions are high and AMP and/or ADP are low. are high.
Phosphofructokinase
AlP Fructose-1,6-
bisphosphatase
ADP p.I
Fructose-1,6-bisphosphale
Fructoa.-S- Fructoa.-1,S-
ATP + phoaphat. -+ blaphoaphat. + ADP
Fructo ..-1,S- Fructoa.-S-

blaphoaphat. -+ phoaphat. + PI
SUM:
ATP PI + ADP
Figure 18.4 Futile cycle resulting from the simultaneous activity of phosphofructokinase and fructose-1,6-
hisphosphatase. The net effect is the hydrolysis of ATP to ADP and the release of large amounts of ener-
gy as heat.
The purpose of gluconeogenesis is to syn- enzymes occurs through their interaction with
thesize sugars and through them the more fructose-2,6-bisphosphate (see Chapter 22).
complex carbohydrates which act as energy
18.4.2 THE FATE OF FRUCTOSE-6-PHOSPHATE
reservoirs. Cells can store energy only if there
is energy to spare. The process of gluconeoge- In some circumstances the gluconeogenesis
nesis should act only when ATP levels are pathway stops with fructose which is found in
high and AMP and/or ADP levels are low. It a number of cells; for instance mammalian
appears that AMP is the principal inhibitor of semen has high levels of this sugar. Fructose is
fructose-l,6-bisphosphatase, which is stimu- liberated from its phosphate under the influ-
lated by citrate. Hormonal control of these ence of a phosphatase.
The synthesis of complex carbohydrates 243
Alternatively, if fructose is to be further CoA (Figure 18.5). Firstly, carboxylation under
processed the enzyme phosphoglucose iso- the influence of propionyl-CoA carboxylase
merase, which takes part in both glycolysis leads to the formation of methylmalonyl-CoA.
and gluconeogenesis, changes fructose-6- The reaction mechanism is very similar to one
phosphate into glucose-6-phosphate. of the first stages in fatty acid synthesis (see
Chapter 19) where CO 2 is added to acetyl-CoA
to produce malonyl-CoA (acetyl-CoA carboxy-
18.5 THE UTILIZATION OF GLUCOSE-6-
lase). The reaction, which is irreversible,
PHOSPHATE
requires energy in the form of ATP, and uses
In liver and kidney, glucose is produced in its biotin as its coenzyme.
free form for export to other tissues. The The methylmalonyl-CoA formed is
enzyme responsible, glucose-6-phosphatase, rearranged to form succinyl-CoA by the
simply hydrolyses the phosphate group to enzyme methylmalonyl-CoA mutase. This
yield the free sugar and an orthophosphate enzyme is unusual because it is one of very
group. The standard free energy change is few that use vitamin B12 as a cofactor. An effect
large and negative. In glycolysis the equiva- of cobalt deficiency in ruminants is to interfere
lent step, using hexokinase, involves the trans- with the operation of this pathway, which is of
fer of orthophosphate from ATP - the free critical importance to the ruminant. Succinyl-
energy change is also large and negative. Once CoA is used to provide phosphoenolpyruvate,
again this raises the possibility of a futile cycle via oxaloacetate, which is then used to synthe-
that achieves only the hydrolysis of ATP to size glucose-6-phosphate.
ADP and orthophosphate.
18.7 THE SYNTHESIS OF COMPLEX
18.6 GLUCONEOGENESIS FROM CARBOHYDRATES
PROPIONATE
18.7.1 DISACCHARIDES
One pathway which is of outstanding impor-
tance in animal agriculture is the one which Many disaccharides are found in nature.
provides most of the glucose for ruminant Some, such as maltose, exist more commonly
animals. In non-ruminants the major energy as breakdown products of polysaccharides.
supply is in the form of glucose; in ruminants But two compounds are outstanding in their
this role is undertaken by a group of volatile economic importance in agriculture: in ani-
fatty acids (VFAs). Despite the importance of mals, lactose is formed in the lactating mam-
the VFAs in the nutrition of ruminants, the mary gland from galactose and glucose; in
animals still have an absolute need for glu- plants, sucrose, composed of fructose and glu-
cose for a number of important functions cose, is of great commercial importance.
such as supplying energy to the brain and
nervous tissues. The importance of glucose
Lactose synthesis
rises steeply during lactation (see Chapter
31). Only one of the major VFAs, propionate, In the dairy cow, lactose synthesis is a large-
is capable of serving as a precursor for the scale operation. A high-yielding cow (produc-
synthesis of glucose. ing, say, 50 kg milk per day) will secrete 2 kg
The first stage of the pathway, the conver- lactose each day and in a whole lactation of 10
sion of propionate into propionyl-CoA, is com- months will produce nearly half a tonne. This
mon to the metabolism of all free fatty acids gives some indication of the massive strain
(Chapter 13). Thus, the only unusual steps are that lactation imposes on the cow's biochemi-
those that convert propionate into succinyl- cal resources.
Propionate
HSc0t:ATP
AMP
+PP,
o~
~CSCoA
I Propionyl CoA
CH 2
I
CH3 co~ATP
Biotin
AMP
+PP, O~
Methyl malonyl CoA ,
~c SCoA
o~
~CSCoA
1
lvltamln B12
H-C- CH 3
as coenzyme
I
COOH
I
CH 2
C:
I Succinyl CoA
H2
9
COOH
CoASH
+ DP
P,
GTP
r--
Succinate
FAD
~"----. FADH2
Oxaloacetate
F=::p+co,
Phosphoenolpyruvate
•..
Glucose
•
Figure 18.5 Glucogenesis from propionate. Essentially, these reactions are the reverse of those used by
microorganisms in the formation of propionate. A shortage of vitamin B12 or of the cobalt required to syn-
thesize it has serious consequences in sheep which rely greatly on this pathway for their supply of glucose.
The mammary gland takes up glucose from every molecule of lactose produced the path-
blood for lactose synthesis. In outline, for way needs two molecules of glucose, half of
which is converted into galactose before being albumin. This protein links itself to galactosyl
used. The first part of the pathway therefore transferase and modifies its action so that its
consists of the production of galactose in an specificity changes. Instead of N-acetyl-o-glu-
activated form. cosamine as its principal substrate, it now uses
As with almost all intracellular reactions of glucose. Once the enzyme is bound to a-lactal-
glucose, the first step is phosphorylation at the bumin, the complex is known as lactose syn-
6 position under the influence of hexokinase. thase.
glucose + ATP-> UDP-galactose + glucose ~ lactose + UDP
glucose-6-phosphate + ADP
This reaction is unusual for another reason
The next step is to move the phosphate - because the second molecule of glucose is
group from the 6 position to the 1 position incorporated into lactose without first being
under the influence of phosphoglucomutase. phosphorylated. In almost all other circum-
The reaction is freely reversible and is also stances, both in higher plants and in animals,
involved in the metabolism of many other glucose is phosphorylated as soon as it enters
sugars. Glucose-I-phosphate also has a role in cells. The overall sequence of reaction in lac-
the oxidation of galactose via glucose, and is a tose synthesis is shown in Figure 18.6.
key intermediate in the synthesis of starch,
glycogen and cellulose.
The synthesis of sucrose
glucose-6-phosphate ~ glucose-I-phosphate
Sucrose is usually found as an end product of
Glucose-I-phosphate is then activated by photosynthesis, indeed its major commercial
conversion to UDP-glucose which uses the source is sugarcane which is the most efficient
enzyme UDP-glucose pyrophosphorylase. photosynthetic plant known. Once again the
starting material for the pathway is UDP-glu-
glucose-I-phosphate + UTP ~
cose, formed under the influence of UDP-glu-
UDP-glucose + PPj
cose pyrophosphorylase. The glucose unit is
The enzyme UDP-glucose-4-epimerase then transferred to the 1 carbon of fructose-6-
catalyses the transformation of glucose to phosphate, yielding sucrose-6-phosphate:
galactose by changing the orientation of the
UDP-glucose + fructose-6-phosphate ->
groups on carbon 4.
sucrose-6-phosphate + UDP
UDP-glucose ~ UDP-galactose
The reaction is catalysed by sucrose phos-
The next step in the pathway involves an phate synthase, a large (360-400 kDa)
enzyme complex with unusual properties. In a allosteric enzyme which is inhibited by
whole range of species, the enzyme galactosyl sucrose. The sucrose-6-phosphate is then
transferase is bound to the Golgi bodies. This hydrolysed to liberate the sucrose:
normally catalyses the transfer of a galactose
sucrose-6-phosphate -> sucrose + Pj
group from UDP-galactose to a sugar deriva-
tive known as N-acetyl-o-glucosamine (see The relationship between these steps is
Figure 3.5f) but not to glucose. This reaction is shown in Figure 18.7.
a normal component of the process for the Despite the role of sucrose as the ultimate
synthesis of glycoproteins. During pregnancy, end point of photosynthesis, the enzymes
when the mammary gland is developing, it UDP-glucose pyrophosphorylase and sucrose
starts to produce proteins that are characteris- phosphate synthase are found not in the
tic of milk and are not found under other cir- chloroplast but in the cytoplasm. The transfer
cumstances. Amongst these proteins is a-I act- between the pathways of photosynthesis and
Glucose 6-phosphate
ADP
/
Glucose 1-phosphate
Glucose
AlP
,. ,
,,
,
UDP-glucose \2 Glucose \
/
/
UDp-ga lactose
..
/
Glucose
Lactose synthase
UDP
ILactose I
Figure 18.6 Overall pathway for the synthesis of lactose from two molecules of glucose. One of these
enters the pathway in the form of glucose-6-phosphate but, unusually, the pathway also uses one mole-
cule of glucose directly.
sugar formation takes place through the com- Approximately every tenth glucose unit there
pound 3-phosphoglycerate. This is produced are branching points at which there is also an
in the chloroplast and actively transferred out «-1,6 linkage to a second chain of glucose
by a membrane-bound system that simultane- residues. A separate, branching enzyme is
ously carries phosphate in. responsible for the formation of these branch-
es.
18.7.2 SYNTHESIS OF POLYSACCHARIDES
Starch synthesis
Glycogen synthesis
Synthesis of starch takes place in two different
The starting material for glycogen synthesis is locations within plants: the first is in the
UDP-glucose, from which glucose is added chloroplasts of green tissues, and the second is
sequentially to the growing saccharide chain. in specialized intracellular organelles, the
The enzyme catalysing the reaction is glyco- amyloplasts, which are found in reserve tis-
gen synthetase/which elongates the chain by sues such as seeds and roots. Amyloplasts
adding glucose groups at carbon 4 of the last resemble chloroplasts in the double-layer form
glucose in the chain (<<-1,4 bonding). of their surrounding membranes, but they do
Glucose 6-phosphate
/
Glucose 1-phosphate
UDP-glucose Fructose 6-phosphate
UDP
Sucrose 6-phosphate
H'0:=:i
PI
Sucrose
Figure 18.7 Pathway for the synthesis of sucrose. Sucrose is actually produced in the form of its phos-
phate which is then dephosphorylated to yield free sugar.
not contain any of the specialized enzymes of action of a branching enzyme. Starch synthase
photosynthesis. Starch synthesis takes place has both a catalytic and a controlling function
by a mechanism that is broadly similar to that in starch synthesis - it is activated by 3-phos-
of glycogen. In essence, activated glucose is phoglycerate and inhibited by high concentra-
formed by a pyrophosphorylase enzyme and tions of orthophosphate. This is easily
this is used to elongate the growing chain of explained in chloroplasts because high levels of
sugar units. The difference between this reac- 3-phosphoglycerate are to be expected in the
tion sequence and that for other polysaccha- light when photosynthesis is at its maximum.
rides is that it uses ATP instead of UDP. At the same time, the levels of Pj will be low
due to the fact that most phosphate will be
ATP + a-glucose-l-phosphate ~
incorporated into ATP. This ensures high rates
ADP-glucose + PPj
of starch synthesis in chloroplasts when 3-
The enzyme is ADP-glucose pyrophospho- phosphoglycerate is formed in photosynthesis.
rylase. It is easy to understand the role of 3-phos-
Starch synthase then catalyses the transfer phoglycerate (a direct product of photosyn-
of the sugar from ADP-glucose to the growing thesis) in chloroplasts, but its importance in
polysaccharide. On its own, this enzyme is amyloplasts is not as obvious. The main pre-
responsible for the formation of amylose; the cursor for starch synthesis in amyloplasts is
synthesis of amylopectin also requires the sucrose, previously synthesized in the chloro-
plasts. Two mechanisms have been suggested enzymes that elongate the chains function by
for sucrose utilization. The first is the hydroly- adding UDP-glucose units in a similar way to
sis of sucrose to glucose and fructose by inver- glycogen synthesis. The pathway first involves
tase. Both glucose and fructose can then be the formation of UDP-glucose by a pyrophos-
used as precursors for the formation of glu- phorylase reaction but, unlike the situation in
cose-I-phosphate. The second is a reversal of glycogen synthesis, the sugar is in the ~ orien-
the reaction catalysed by sucrose synthase, tation.
yielding UDP-glucose.
sucrose + UDP ~ UDP-glucose + fructose
UTP + ~-glucose-l-phosphate ~ UDP-
The UDP-glucose is then hydrolysed to lib-
erate glucose-I-phosphate.
glucose + PPi
This activated sugar is then used as the unit
UDP-glucose + Hp ~
for elongating the polysaccharide chain. One
UMP + glucose-I-phosphate
of the main problems in studying cellulose
UDP-glucose, ADP-glucose and glucose-l- synthesis has been the fact that it is not possi-
phosphate cannot enter the amyloplast with- ble to isolate the separate parts of the path-
out modification. Sugar for starch synthesis way and still retain high levels of the right
thus enters the amyloplast as triosephos- sort of activity. Cellulose is a ~-1,4 chain, but
phate. The transport of 3-phosphoglycerate is in isolated systems the enzymes of cellulose
active and involves phosphate groups being synthesis produce a different polysaccharide
simultaneously pumped out of the organelle called callose, in which the glucose units are
(Figure 18.8). arranged ~-1,3. Callose is naturally produced
The rationale can now be seen for the acti- when plants and trees are physically dam-
vation of starch synthase by high concentra- aged. The enzyme responsible for elongation
tions of 3-phosphoglycerate and low levels of is cellulose synthase which is located within
Pi· the plasma membrane of the cells. Cellulose
Within the amyloplast, the enzymes of synthase molecules are found in groups
glucogenesis take over and resynthesize glu- arranged in a rosette form. Electron micro-
cose-6-phosphate. Glucose-6-phosphate is scopy following fracture of the cell wall has
converted to glucose-I-phosphate, at which revealed growing microfibrils of cellulose
point it is available as a substrate for the attached to the enzyme complexes. One theo-
pyrophosphorylase reaction. ry to explain the fact that intact cells produce
In the synthesis of amylopectin there is an cellulose, whereas damaged ones form cal-
enzyme which is able to transfer an oligosac- lose, involves a modifier protein. Cellulose-
charide unit onto the 6-carbon of a limited producing cells contain a relatively small (18
number of glucoses in the polysaccharide kDa) protein which appears to be needed for
chain. These a-l,6linkages form the branching activity by the membrane-bound cellulose
points which lead to the characteristic struc- synthase (see Figure 18.9).
ture of amylopectin. If the cells are intact they will have low intra-
cellular levels of calcium ions and a negative
electrical potential across the membrane. Under
Cellulose synthesis
these conditions the enzyme complex will syn-
The main purpose of cellulose is a structural thesize cellulose. However once cells are dam-
one, and for that reason its synthesis takes aged, higher levels of calcium will be encoun-
place in such a way that the cellulose is pro- tered near the enzyme, the electrical potential
duced ready for use in microfibrils. The across the membrane will be lost and the solu-
Cytoplasm
STARCH
SUCROSE
\
<gluco.e)n.1
•
I ADP)
glucose-6-phosphat. /<9IUCOee)n
1
ADPglucos.
\
glucos.-'-phosphate
fructos.-6-phoaphat.
t
\
glucoa.-6-phospha••
i
fruclo •• -6-phosphate
2 3-GPA
1
Figure 18.8 Pathway for the transport of sugars from sucrose into the amyloplast for starch synthesis.
The major metabolite absorbed by the amyloplast is 3-phosphoglycerate.
ble 18-kDa protein will be dissociated from the interesting to compare the activity of this mod-
enzyme complex. In the absence of the modifi- ifier protein in plants with the animal protein
er protein and under the conditions of a dam- (a-lactalbumin) which has a somewhat similar
aged cell, the enzyme produces callose. It is role in lactose synthesis.
Insolubl. glucosyl fr.ns'.r.s. with
18 kd.1 modlfl.r prof.'n UDP
UDP-B-glucos.
+ • +
(8-1,4-gluco •• chaln)n+l
(8-1,4-gluco •• chaln)n CELLULOSE
Insoluble glucosyl trans'erase

- no 1B kdal modifier proteIn UDP
UDP-B-gluco ••
+
+ HIgh Ca 2 + & dissipated M (B-1,3-glucos. chaln)n+l
(8-1,3-gluco .. chain)n
CALLOSE
Figure 18.9 Synthesis of cellulose and callose. Normally the conditions favour cellulose synthesis. When
the plant is damaged the changes in Ca2+ ions and in membrane electrical potential lead to callose syn-
thesis. ~ 'I' is the electrical potential across the plasma membrane.
SYNTHESIS OF FATTY ACIDS AND LIPIDS 19

19.2 Tissue and subcellular location of fatty 252
acid synthesis
19.3 Source of the primary substrate - acetate 252
19.4 Production of malonyl-CoA 253
19.4.1 Animals 254
19.4.2 Plants 256
19.4.3 Bacteria 256
19.5 Synthesis of long-chain saturated fatty 256
acids from acetyl-Co A and malonyl-CoA
19.5.1 Acyl carrier protein and its function 256
19.5.2 The reactions of fatty acid synthesis 257
19.5.3 Chain length specificity of fatty acid 259
synthetases
19.5.4 Synthesis of branched-chain fatty acids 260
19.5.5 Release of fatty acids from fatty acid 261
synthetase
19.6 Fatty acid elongation 261
19.7 Formation of unsaturated fatty acids 262
19.7.1 Desaturation of fatty acids in animals 262
19.7.2 Desaturation of fatty acids in plants 263
19.7.3 Essential fatty acids 263
19.8 Synthesis of triacylglycerols 265
19.8.1 The 2-monoacylglycerol pathway 265
19.8.2 The glycerol-3-phosphate pathway 266
19.8.3 Triacylglycerol synthesis in plants 267
19.9 Phospholipids 268
19.10 Glycolipid synthesis 268
19.11 Synthesis of sphingolipids 270
19.12 Biosynthesis of terpenes and sterols 271
19.12.1 Synthesis of mevalonic acid 271
19.12.2 Conversion of mevalonic acid to 273
squalene
19.1 INTRODUCTION acids and lipids they require. In higher plants

and animals, diversification of cells and evo-
All cells contain lipids, most of which contain lution of tissues have occurred, resulting in
fatty acids. Unicellular organisms possess all the development of tissues with specialized
of the biochemical machinery necessary to functions. In the context of fatty acid and lipid
synthesize their cellular components and are, synthesis this is more apparent in animals
therefore, capable of synthesizing the fatty than in plants.
252 Synthesis of fatty acids and lipids
In animals, de novo fatty acid synthesis pose tissue and mammary tissue. Liver is the
occurs mainly in adipose tissue, liver and site of 90% of fatty acid synthesis in avian
mammary tissue. Fatty acids may be moved species, whereas in ruminants the major site of
around the body from the site of synthesis in fatty acid synthesis (90%) is the adipose tissue,
the form of transport lipids, mainly phospho- and liver makes only a minor contribution to
lipids and triacylglycerols, but also to some the de novo synthesis of fatty acids. In non-
extent as non-esterified fatty acids. Certain tis- ruminant mammals, both the liver and adi-
sues are specialized for the production of pose tissue make a contribution to fatty acid
complex lipids. For example: synthesis. The relative contributions of the
two tissues differ from species to species. In
• intestinal mucosa transforms the products
the lactating animal, mammary tissue is a very
of lipid digestion into triacylglycerols and
active site of fatty acid synthesis. For example,
phospholipids;
a high-yielding dairy cow may secrete as
• liver incorporates newly synthesized and
much as 1.25 kg milk fat per day, which repre-
absorbed fatty acids mainly into phospho-
sents de novo synthesis within the mammary
lipids which are secreted into the blood as
tissue of 400-450 g fatty acid per day.
lipoproteins;
In plants, most tissues are capable of syn-
• adipose tissue synthesizes predominantly
thesizing fatty acids. Certain tissues are par-
triacylglycerols which are stored in fat
ticularly active in fatty acid synthesis, for
droplets and mobilized when required;
example, the seeds of rape, linseed and soy-
• mammary tissue is very active in the synthe-
abean. In animals, de novo fatty acid synthesis
sis of milk triacylglycerols during lactation.
takes place in the cytoplasm; in plants it
Most plants do not store large quantities of appears that the chloroplast is the subcellular
lipids, with the exception of some oilseeds. organelle responsible for the synthesis of
Most lipids in plants, therefore, have a struc- fatty acids in mesophyll cells. In other plant
tural role as components of membranes and cells, particularly those in seeds, the subcellu-
are synthesized in situ in each cell. For this rea- lar location of fatty acid synthesis is less clear.
son, plants do not transport fatty acids and
complex lipids between their tissues. From an
19.3 SOURCE OF THE PRIMARY SUBSTRATE-
agricultural standpoint the most important
ACETATE
plant tissues involved in lipid synthesis are the
seeds. In some plant species, seeds produce Acetate is the basic two-carbon unit from
large quantities of triacylglycerols which are which fatty acids are synthesized. Prior to
mobilized during germination. A large agricul- incorporation into fatty acids it must first be
tural and food industry has developed around converted to acetyl-CoA. As acetyl-CoA is pro-
the extraction and utilization of lipids from duced in large quantities from pyruvate in the
oilseeds. Recent developments in biotechnolo- mitochondria, there would appear to be no
gy allow the manipulation of the fatty acid shortage of this important building block.
composition of oilseed triacylglycerols. This However, the production of acetyl-CoA from
offers exciting possibilities for the use of genet- pyruvate and its utilization for fatty acid
ically manipulated oilseed products for nutri- synthesis take place in different subcellular
tional and industrial purposes. compartments: production takes place in the
mitochondrial matrix, and fatty acid synthesis
in the cytoplasm. Because the inner mitochon-
19.2 TISSUE AND SUBCELLULAR LOCATION
drial membrane is impermeable to coenzyme
OF FATTY ACID SYNTHESIS
A and its derivatives, mitochondrial acetyl-
In animals, the most important tissues CoA is not readily available for fatty acid syn-
involved in fatty acid synthesis are liver, adi- thesis in the cytoplasmic compartment.
Production of malonyl-eoA 253
In non-ruminant animals, glucose is the activity in ruminant adipose and mammary
main source of the acetate required for fatty tissue than in tissues of non-ruminant animals.
acid synthesis. Pyruvate, produced by the oxi- Due to the effects of rumen fermentation of
dation of glucose via the glycolytic pathway, is dietary carbohydrate, little digestible carbohy-
transported into the mitochondrial matrix. drate reaches the small intestine and therefore
Here pyruvate dehydrogenase converts it to absorption of glucose from the digestive tract
acetyl-CoA which enters the TCA cycle where it is small. It would, therefore, be wasteful for
condenses with oxaloacetate to produce citrate ruminants to use glucose, produced by gluco-
(Chapter 11). In most tissues citrate is oxidized neogenesis, for fatty acid synthesis, when
via the TCA cycle. However, in those tissues there is a plentiful supply of acetate. Although
where fatty acid synthesis takes place, some of in ruminants citrate can still leave the mito-
the citrate is diverted to the cytoplasm to serve chondria, the conversion of glucose to cyto-
as a source of acetyl-CoA. In the cytoplasm, cit- plasmic acetyl-CoA via citrate is prevented in
rate is split into oxaloacetate and acetyl-CoA by ruminants by the extremely low activities of
the enzyme ATP-citrate lyase, sometimes also ATP-citrate lyase and malic enzyme. Instead of
called the citrate cleavage enzyme. This acetyl- being cleaved to acetyl-CoA and oxaloacetate,
CoA can then be used for fatty acid synthesis. this citrate is converted to isocitrate by aconi-
The oxaloacetate produced by ATP-citrate tase, and then acted upon by a cytoplasmic
lyase can be recycled to the mitochondria via NADP-dependent isocitrate dehydrogenase
pyruvate. This is achieved by the action of a which produces the NADPH needed for fatty
further two enzymes, NAD-dependent malate acid synthesis. The a-ketoglutarate also pro-
dehydrogenase, which converts oxaloacetate duced can be translocated back into the mito-
into malate, and NADP-dependent malate chondrial matrix (Figure 19.2). It is estimated
dehydrogenase (the malic enzyme) which that in ruminant mammary tissue, and proba-
catalyses the oxidative decarboxylation of bly in adipose tissue, NADP-dependent isoci-
malate to produce pyruvate. This pyruvate trate dehydrogenase can produce similar
can re-enter the mitochondrial matrix. The net amounts of NADPH to those produced by
effect of this cycle of reactions is the transport malic enzyme in non-ruminants.
of acetyl-CoA from the mitochondrial matrix In plants, acetyl-CoA is produced from glu-
to the cytoplasm (Figure 19.1). cose via the glycolytic pathway in non-photo-
Another product of the NADP-dependent synthetic tissues. In photosynthetic tissues,
malate dehydrogenase reaction is NADPH, pyruvate can also be produced from glycer-
which is required for fatty acid synthesis. In aldehyde-3-phosphate synthesized by the
theory this reaction could provide 50% of the Calvin cycle. In addition, there is an active
NADPH required for fatty acid synthesis if all acetyl-CoA synthetase in chloroplasts which is
the oxaloacetate produced by ATP-citrate capable of activating any acetate generated
lyase were converted to pyruvate. It is likely, outside the chloroplast.
however, that between 40 and 50% of the
NADPH needed for fatty acid synthesis comes
19.4 PRODUCTION OF MALONYL-COA
via this route, the remaining 50-60% coming
from the oxidation of glucose via the pentose In addition to acetyl-CoA, malonyl-CoA is
phosphate pathway. also an essential substrate for fatty acid syn-
In ruminants, the fermentation of carbohy- thesis and is produced by the carboxylation
drate in the rumen produces large quantities of acetyl-CoA, catalysed by the enzyme
of acetate which is transported to the tissues acetyl-CoA carboxylase. This is a complex
via the bloodstream. This acetate is converted enzyme that occurs in animals, bacteria and
to acetyl-CoA by the action of acetyl-CoA syn- plants. Its properties are different in these
thetase, which exhibits considerably higher three different types of organism.
C
Glucose
I Fatty acids
I - - . . . - ..
(Pentose 4 - - - " , , ? , , - - - NAOp·
I phosphate
'pathway"
I " __ __ ~
-...---II--......~NAOPH + H'
I
NAOPH + H+
+ 2
~3
"'~I-\....--''''''----;~------Malate
CO )NAOP+
Pyruvate
\NAD+
Pyruvate
\
®\NADH+H+
O~
Acetyl-CoA
OxAc, Jtv l CoA

-
Citrate - - - - t t - - - - -.... Citrate

TCACycle
CYTOPLASM
IMITOCHONDRIAL MATRIX
Figure 19.1 Production of NADPH and cytoplasmic acetyl-eoA for fatty acid synthesis in non-ruminant
tissues. Enzymes indicated are: 1, ATP-citrate lyase; 2, NAD-dependent malate dehydrogenase; 3, NADP-
dependent malate dehydrogenase (the malic enzyme). OxAc = oxaloacetate.
19.4.1 ANIMALS attached to BCCP. Secondly, carboxyltrans-

ferase catalyses the transfer of the carboxyl
In animals, the carboxylase is an allosteric group from biotin to acetyl-CoA, produdng
enzyme. It exists in two forms, as an inactive malonyl-CoA. BCCP carries the biotin mole-
monomer and as an active polymer which cule. The overall reaction is shown in Figure
consists of a linear chain containing approxi- 19.3.
mately 20 monomer units. The monomer unit The control of activity of acetyl-CoA car-
of acetyl-CoA carboxylase is a 250-kDa multi- boxylase is complex. Both acute (short-term)
functional protein which has three distinct and chronic (long-term) regulation appear to
domains. Two of these, biotin carboxylase and occur, and nutritional and hormonal status
carboxyltransferase, have enzymic activities; have a significant impact on the activity of the
the third acts as a carrier protein, biotin car- enzyme. Polymerization of the inactive
boxyl carrier protein (BCCP). Each monomer monomer units to the active polymer is stimu-
also contains one molecule of biotin and a reg- lated by dtrate. Citrate acts as an indicator of
ulatory allosteric site. energy supply and in this way directs excess
The production of malonyl-CoA is the sum carbon towards storage as fatty adds. The end
of two intermediate reactions. Firstly, biotin products of fatty add synthesis, fatty acyl-
carboxylase catalyses the carboxylation of biotin CoAs, cause depolymerization of the enzyme,
Production of malonyl-CoA 255
Glucose Fatty acids
1--"'-...
I
( Pentose
I phosphate
~ ..p~':f! - '
C +
NADP ....- - -
NADPH + H+
I
I CoA
+ Acetate '>.... .. Acetyl-CoA
®
Pyruvate
~
."
:r:
+
:r:+
Pyruvate ~
"
" +
Acetyl-CoA
OxAc~itrate _ _ _....-....- Citrate
TCACycie . ,
lsocitrate
lsocitrate
J
lutarate
o
«-ketoglutarate
MITOCHONDRIAL MATRIX I CYTOPLASM
Figure 19.2 Production of NADPH and cytoplasmic acetyl-CoA for fatty acid synthesis in ruminant tis-
sues. Enzymes indicated are: 1, NADP-dependent isocitrate dehydrogenase; 2, acetyl-CoA synthetase.
OxAc = oxaloacetate.
thus decreasing its activity and reducing the and the catecholamines, which decrease the
supply of substrates for fatty acid synthesis. acetyl-CoA carboxylase activity, cause phos-
In addition, the enzyme is regulated by phorylation via an increase in the intracellular
phosphorylation and dephosphorylation concentration of cAMP and the subsequent
caused by glucagon, the catecholamines and activation of protein kinases. Insulin, which
insulin. Phosphorylation decreases the activity has lipogenic effects, also promotes phospho-
of the enzyme. The detailed mechanisms of rylation: its main effects may be via activation
the mode of action of these hormones are still of phosphatases which decrease the phospho-
unclear. At present it is thought that glucagon rylation state of the enzyme.
acetyl-CoA
carboxylase
Acetyl-CoA r ~
ATP
rI."
CH 3COSCoA + HC03- --7.-:::::-B-io-t;:"":"'n'~-". «HzCOSCoA
ADP + PI
COOH
Malonyl-CoA
Figure 19.3 Overall reaction catalysed by acetyl-CoA carboxylase.

19.4.2 PLANTS place on a complex enzyme called fatty acid
synthetase. Fatty acid synthetases have been
Acetyl-CoA carboxylase in plants is similar in
characterized into three groups known as
structure to that in other eukaryotic cells. The
types I, II and III. Types I and II are involved
enzyme is located in the chloroplasts in leaf tis-
in the de novo synthesis of fatty acids; type III is
~ue a~d in flastids in seeds. Unlike the enzyme
mammal tissues, the plant enzyme is not acti- in:olved in. the elongation of existing fatty
aC1ds and w1ll be considered later. The main
vated by citrate; instead, small changes in stro-
mal pH or Mg2+ and K+ concentration can feature that distinguishes the type I and II syn-
thetases is the functional organisation of their
markedly affect enzyme activity. The enzyme
constituent proteins.
is also regulated by a heat-stable factor found
Type I synthetases are found in animals,
in leaves and is influenced by the ratio of ADP
yeasts and some bacteria. These enzymes are
to ATP. High ATP levels activate the enzyme.
large multifunctional proteins containing a
~umber of enzyme activities on one polypep-
19.4.3 BACTERIA tide chain. In addition, they contain a carrier
protein called acyl carrier protein or ACP.
In bacteria, acetyl-CoA carboxylase is active in
Type II synthetases occur in most bacterial
its monomeric form and there is no evidence
and plant tissues. The functional enzyme is a
that it polymerizes.
multi enzyme complex made up of a number
. Th~ major difference between the enzyme
of individual enzymes and ACP loosely bound
mammals and bacteria is that in bacteria the
together. The individual enzymes can be iso-
monomer readily dissociates into its three con-
lated in active form; this cannot be done with
stituent proteins: biotin carboxylase, biotin car-
type I synthetases.
boxyl carrier protein, and carboxyltransferase.
Despite the differences in the structural
Thus the monomer is not a multifunctional
organisation of the fatty acid synthetase
protein but consists of individual proteins
enzyme in different organisms, the overall
which come together to form the monomer.
sequence of reactions that takes place is similar.
The bacterial enzyme is controlled differ-
ently to the animal enzyme. Its activity is
reduced by the presence of the nucleosides 19.5.1 ACYL CARRIER PROTEIN AND ITS
guanosine-3' -diphosphate,S' -diphosphate FUNCTION
(ppGpp) and guanosine-3' -diphosphate, 5'-
A distinctive feature of fatty acid synthesis is
triphosphate (pppGpp). These guanosine
that the acyl intermediates are attached to a
nucleosides are unique to bacteria and are pro-
low-molecular-weight protein, acyl carrier
duced when they are not growing rapidly. In
protein (ACP). This protein, which is an inte-
bacteria, the synthesis of fatty acids is mainly
gral part of all fatty acid synthetases, has 77
to meet the requirement for membrane lipids,
a~i~o acids. The functional part of the pro-
so it is logical that fatty acid synthesis should
tem 1S a molecule of 4' phosphopantotheine
be reduced in conditions when bacteria are
~hich is attached to a serine residue at posi-
not growing and when new membranes are
hon 36. The 4' phosphopantotheine, which
not being synthesized.
has a terminal SH group to which the acyl
intermediates are attached by a thio-ester
19.5 SYNTHESIS OF LONG-CHAIN
bond, is identical to that found in CoA. ACP
SATURATED FATTY ACIDS FROM ACETYL-
acts as a flexible arm which functions to
COA AND MALONYL-COA
transfer the fatty acid intermediates from one
The synthesis of saturated straight-chain fatty site of enzyme activity to the next (Figure
acids from acetyl-CoA and malonyl-CoA takes 19.4).
Synthesis of long-chain saturated fatty acids from acetyl-CoA and malonyl-CoA 257
Coenzyme A
o 0 ?H CH 0 0
II " I 3 II"
HS-CH-CH-N-C-CH-CH-N-C-CH-C-CH-O-P-O-P-O-CH
2 2H 2 2H I 2 I I 2
I C~ ~ 6
o OH
r
4'.Phosphopantotheine
I _
o=P-o
1-
o
1
o
II
0 ?H CH
II I 3
0
II"
I 0
HS-CH-CH-N-C-CH-CH-N-C-CH-C-CH-O-P-O-P-O-Ser
2 2H 2 2H I 2 I I
CH --
3 0 0
Acyl Carrier Protein (ACP)
Figure 19.4 Structures of coenzyme A and acyl carrier protein (ACP).
19.5.2 THE REACTIONS OF FATTY ACID Once the acetyl residue has been trans-
SYNTHESIS ferred to l3-ketoacyl ACP synthase, the ACP
The overall scheme of de novo fatty acid syn- molecule is free to accept a malonyl residue.
thesis is shown in Figure 19.5. The first step in This reaction is catalysed by the enzyme mal-
the synthesis of a fatty acid is the attachment of onyl transacylase (reaction 2)
the substrates to the enzyme. Initially acetyl- With both substrates in position on the
CoA reacts with the sulphydryl group of ACP enzyme, there then follows a condensation
to form acetyl-ACP (reaction 1a). This reaction reaction between the malonyl-ACP and the
is catalysed by the enzyme acetyl-transacylase, acetyl synthase. This yields the keto interme-
and acetate is termed the primer molecule. diate, acetoacetyl-ACP, and is catalysed by the
The acetyl residue does not remain on the enzyme l3-ketoacyl ACP synthase (reaction 3).
ACP, however, as ACP is required for the During the condensation reaction, malonyl-
attachment of a malonyl residue transferred ACP is decarboxylated and carbon dioxide is
from malonyl-CoA. Thus the acetyl residue is released. Isotopic studies have shown that this
transferred from ACP to a sulphydryl group is the same CO 2 that is added during the
on the enzyme l3-ketoacyl ACP synthase (reac- acetyl-CoA carboxylase reaction, hence there
tion 1b). is no net incorporation of radioisotope into
REACTION SEQUENCE OF FATTY ACID SYNTHESIS
(1) Priming Reaction

(a) Enzyme:- acetyl transacylase
CH3CO-S-CoA + ACP-SH ~.===~h CH 3CO-S-ACP + CoA-SH
(acetyl-CoA) (acetyl-A CP)
(b) Enzyme:- j3-ketoacyl-ACP synthase

CH 3CO-S-ACP + Synthase-SH ~.===~. CH3CO-S-synthase + ACP-SH
(acetyl-synthase)
(2) Malonyl Transfer
Enzyme:- malonyl transacylase
CH 2CO-S-CoA + ACP-SH T,==~. CH 2CO-S-ACP + CoA-SH

I I
COOH COOH
(malonyl-CoA) (malonyl-ACP)
(3) Condensation Reaction

Enzyme:- j3-ketoacyl-ACP synthase
CH 3CO-S-synthase + CH 2CO-S-ACP ~ CH3COCH 2 CO-S-ACP + CO2

I
COOH (acetoacetyl-ACP)
(4) First Reduction Reaction

Enzyme:- j3-ketoacyl-ACP reductase
CH 3COCH 2CO-S-ACP + NADPH + H+ T.==~h CH 3CHCH 2CO-S-ACP + NADP+

I
OH
(D-3-hydroxybutyryl-S-ACP)
(5) Dehydration Reaction
Enzyme:- enoyl-ACP hydratase
CH 3y HCH2CO-S-ACP T.==~. CH 3CH=CHCO-S-ACP

OH (crotonyl-ACP)
(6) Second Reduction Reaction

Enzyme:- enoyl-ACP reductase
CH 3CH=CHCO-S-ACP + NADPH + H+T.==~ CH 3CH 2CH 2CO-S-ACP + NADP+

(butyryl-ACP)
Figure 19.5 Reaction sequence of fatty acid synthesis. These reactions are required for the production of
the four-carbon fatty acyl intermediate, butyryl-ACP. In the synthesis of palmitoyl-ACP this cycle of reac-
tions is repeated a further six times from priming reaction 1(b). At the start of the second cycle, butyryl-
ACP substitutes for acetyl-ACP in reaction 1(b). In subsequent cycles the product of reaction 6 replaces
acetyl-ACP in reaction 1(b) until the final product, containing 16 carbons, is produced.
Synthesis of long-chain saturated fatty acids from acetyl-CoA and malonyl-CoA 259
fatty acids when tissue preparations are incu- fatty acids increase when ruminants are fed on
bated with [14C]-bicarbonate. diets rich in cereals which increase the molar
The four-carbon intermediate, acetoacetyl- proportion of propionate in the rumen liquor.
ACP, is reduced in a reaction catalysed by the In extreme cases, in sheep, this has been
enzyme J3-ketoacyl-ACP reductase. The shown to lead to unusually high levels of these
hydrogen donor used in this reaction is acids (10-15%) in adipose tissue depots.
NADPH and the product is D-3-hydroxy-
butyrate (reaction 4).
19.5.3 CHAIN LENGTH SPECIFICITY OF FATTY
The next step is the dehydration of the
ACID SYNTHETASES
hydroxyacyl intermediate resulting in the for-
mation of a 6.2-trans enoyl-ACP. This reaction
Plants and animals
is catalysed by the enzyme enoyl-ACP dehy-
dratase (reaction 5). In most species of plants and animals, approx-
The unsaturated intermediate is then imately 90% of the fatty acids produced have
reduced by the addition of hydrogen across 16 carbons, e.g. palmitoyl-ACP. The enzyme
the double bond to yield the saturated inter- responsible for this chain-length specificity is
mediate butyryl-ACP. This reaction is catal- J3-ketoacyl-ACP synthase. At the end of each
ysed by the enzyme enoyl-ACP reductase sequence of four reactions, the growing fatty
(reaction 6). acid is transferred from ACP to the free SH
This reaction completes the process by group of J3-ketoacyl-ACP synthase. This
which the original two-carbon unit, acetyl- enzyme will readily accept a fatty acid con-
ACP, is elongated to a four-carbon unit, taining 14 carbons but will hardly ever accept
butyryl-ACP. The butyryl-ACP becomes the one containing 16 carbons. Thus, the process
starting substrate for the next cycle of reac- of fatty acid synthesis stops once 16 carbons
tions. The butyryl residue is transferred to the have been reached. The newly synthesized
free SH group of J3-ketoacyl-ACP synthase and fatty acid is released from fatty acid synthetase
another malonyl residue is transferred from by acyl-ACP-thioesterase. In animals,
CoA to ACP. The reaction sequence is repeat- thioesterase is an integral part of the syn-
ed to produce a six-carbon intermediate. The thetase complex, whereas in plants it appears
process continues until the growing fatty acid to be a soluble enzyme.
has 16 carbons, e.g. palmitoyl-ACP. The fatty acid composition of milk fat is
In mammary glands, butyrate is also used characterized by the presence of short- and
as a primer molecule, and it has been estimat- medium-chain fatty acids (C4:0-C12:0). As
ed that butyrate carbon contributes approxi- these fatty acids are found in only trace quan-
mately 8% of the total carbon in fatty acids tities in plasma, it is clear that they are synthe-
produced by de novo synthesis in this tissue. sized de novo in mammary tissue. The exact
Odd-numbered fatty acids are produced in mechanism that leads to the termination of
small amounts (0.5-1.0% of the total fatty fatty acid synthesis and release of these fatty
acids) by plant and animal tissues. Ruminant acids is not known for all species of mammals.
fats contain a small but significant quantity of In rat mammary tissue, a second thioesterase
pentadecanoic (C15:0) and heptadecanoic has been identified which catalyses the release
(C17:0) acids. These fatty acids are produced of medium-chain fatty acids. In the goat, the
by the incorporation of the odd-numbered presence of a transacylase is responsible for the
(three-carbon) primer molecule propionyl- transfer of fatty acids from the synthetase com-
CoA, in place of acetyl-CoA. It has been noted plex into milk triacylglycerols. Other factors
that the proportions of these odd-numbered may be important in the control of termination
of fatty acid synthesis in mammary tissue. For branched-chain fatty acids, in which the
example, the chain length of fatty acids syn- methyl substituent is located at various places
thesized in vitro can be influenced by changing along the fatty acid chain. In some cases di-
the relative concentrations of acetyl-CoA and and tri-methyl substituted fatty acids have
malonyl-CoA; the presence of excess acetyl- been identified. In almost all cases the methyl
CoA results in a reduction in the chain length substituent group is located on even-num-
of fatty acids produced. This may be due to bered carbon atoms of the fatty acid chain.
competition between the two substrates for This pattern of methyl substitution provided a
binding sites on the fatty acid synthetase clue to the mechanism by which these fatty
enzyme. However, there is no evidence that acids were synthesized, and it is now accepted
such a mechanism operates in vivo. that they arise as a result of the substitution of
methylmalonyl-CoA for malonyl-CoA during
de novo fatty acid synthesis. A mono-methyl-
Bacteria
substituted fatty acid is produced when only
The chain length of fatty acids produced by one molecule of methylmalonyl-CoA replaces
bacterial fatty acid synthetases varies. For malonyl-CoA and the position of the methyl
example Escherichia coli produces both C16 and substituent depends upon the stage in fatty
C18 saturated and unsaturated fatty acids, acid synthesis at which the replacement
whereas mycobacteria produce saturated fatty occurs. Methylmalonyl-CoA is an intermediate
acids of chain length C16 or C24. in the conversion of propionate to succinate, a
short but important pathway in ruminants
which enables them to use the propionate pro-
19.5.4 SYNTHESIS OF BRANCHED-CHAIN
duced in the rumen to synthesize glucose. The
FATTY ACIDS
feeding of large quantities of cereals which
An interesting feature of fatty acid biosynthesis contain readily fermentable starch leads to the
in bacteria is that most Gram-positive and some rapid production of propionate in excess of
Gram-negative organisms produce mainly the capacity of the liver to metabolize it. This
branched-chain fatty acids of the iso and results in elevated levels of methylmalonic
anteiso series (Chapter 4). These arise as a result acid in the plasma, some of which is excreted
of the use of branched-chain primer molecules in the urine. However, some methylmalonic
in place of acetate (Figure 19.6). These primers acid enters the adipose tissue where it is acti-
are produced by the oxidative deamination of vated and used in fatty acid synthesis. These
branched amino acids. For example, valine is branched-chain fatty acids have a lower melt-
converted to isobutyryl-CoA and isoleucine ing point than saturated fatty acids, and accu-
yields 2-methylbutyryl-CoA. mulation of significant quantities in adipose
Fatty acids of the iso and anteiso series are tissue can result in the production of soft fat.
present in the fat depots of ruminant animals. In water birds a specialized sebaceous
They are found in relatively low proportions gland, the uropygial gland, produces predom-
(0.5-1.5%) and their presence is due to the inantly multi-methyl branched-chain fatty
digestion and absorption of fatty acids synthe- acids, particularly 2,4,6-trimethyldodecanoic
sized by rumen bacteria. When sheep and acid and 2,4,6,8-tetramethyltetradecanoic acid.
goats are fed diets rich in cereals, the propor- This occurs due to the rapid breakdown of
tion of branched-chain fatty acids in their adi- malonyl-CoA in the gland by malonyl-CoA
pose tissue increases. Analysis of these fatty decarboxylase and the use of methylmalonyl-
acids has shown that this is due not only to an CoA as a alternative substrate. These fatty
increase in the iso and anteiso fatty acids, but acids may play an important role in the water-
also to the presence of unusual methyl proofing of plumage.
Fatty acid elongation 261
NH3
CH~H-CH-COOH ___J~__---+. CH~H-C-COOH

I I I II
CH3NH2 CH 3 0
Valine HSCOA~
~C02
CHa-?H-(CH2)n-S-COA .. CH-CH-C-S-CoA
3 I II
CH 3 CH 3 0
Iso fatty acid (malonyl-CoA)n Isobutyryl-CoA
CH-CH-CH-CH-COOH CH-CH-CH-C-COOH
3 2 I I 3 2 I II
CH 3 NH2 CH 3 0
HSCOA~
Isoleucine
~C02
CH-CH-CH-(CH2)n-S-CoA ...- - \ : : - - - - - CH-CH-CH-C-S-CoA
3 2 I 3 2 I II
CH 3 CH 3 0
Anteiso fatty acid (malonyl-CoA)n 2-methylbutyryl-CoA

Figure 19.6 Summary of the synthesis of iso- and anteiso-branched-chain fatty acids from valine and
isoleucine.
19.5.5 RELEASE OF FATTY ACIDS FROM FATTY 19.6 FATTY ACID ELONGATION
ACID SYNTHETASE
De novo synthesis of fatty acids produces main-
Fatty acids may be released from the syn- ly palmitic acid (C16:0). However, analysis of
thetase in a number of forms. In most animals, the fatty acids of plant and animal tissues
fatty acids are released as free fatty acids reveals that there are a large number of fatty
which may then be modified in several ways. acids with more than 16 carbon atoms. Some
For example, they may be elongated, desatu- of these may be supplied in the diet in the case
rated and/or incorporated into other lipids of animals, but both plant and animal tissues
such as triacylglycerols and phospholipids. In have the capacity to elongate fatty acids.
plants and microorganisms, fatty acids may be The enzyme systems involved in fatty acid
released as free acids or may be converted to elongation are sometimes referred to as type III
their CoA esters upon release. In certain fatty acid synthetases, and sometimes as fatty
microorganisms such as E. coli and Euglena gra- acid elongases. The enzymes involved in elon-
cilis the fatty acid is transferred directly from gation appear to be membrane-bound and are
ACP to phosphatidic acid and thereafter into found in two sites in the cell, in mitochondria
triacylglycerols and phospholipids. and the endoplasmic reticulum. The latter is
the main site of fatty acid elongation. The sub- the fatty acid relative to the carboxyl carbon
strates for elongation are fatty acyl-CoAs which (carbon no. 1) where the double bond is
are extended by two carbon units which are inserted.
supplied by malonyl-CoA. Reducing equiva- The desaturation of fatty acids in animal tis-
lents are provided by NADPH. sues is summarized below.
• All desaturases require molecular oxygen
19.7 FORMATION OF UNSATURATED FATTY and a reduced pyridine nucleotide, and
ACIDS catalyse the desaturation of preformed fatty
acids, usually in the form of coenzyme A
The introduction of double bonds into a fatty
esters. There is some evidence that the fatty
acid is called desaturation. There are two
acids in complex lipids can also serve as
processes by which desaturation of fatty acids
desaturase substrates.
occurs. In the first, anaerobic desaturation, an
• For a given desaturase enzyme, the double
unsaturated fatty acid is produced during fatty
bond is always introduced into the methyl-
acid synthesis. This process is of limited impor-
ene chain at a fixed position from the car-
tance and is found only in bacteria, particularly
boxyl group and has the cis configuration.
Eubacteriales. Typical of this process is the pro-
duction of vaccenic acid (~11 cis-octadecenoic e.g. ~9 desaturase always introduces a dou-
acid). The synthesis of this fatty acid occurs ble bond between carbons 9 and 10, not
because the ~z trans-decenoyl-ACP, one of the between 8 and 9 or between 10 and 11.
intermediates produced in reaction 5 of Figure
19.5, is isomerized to form ~2 cis-decenoyl-ACP • When the substrate is a saturated fatty acid,
the first double bond is inserted between
which is not a substrate for enoyl-ACP reduc-
tase. Instead, it acts as the substrate for the con- carbons 9 and 10. Unlike plants, animal
desaturase systems cannot insert double
densation reaction (reaction 3) and is elongated
to an 18-carbon fatty acid. The second, aerobic bonds between carbon 10 and the methyl
desaturation, is found in virtually all organisms. carbon, for example:
In this process, double bonds are introduced e.g. C16:0 --> ~9 C16:1
into preformed fatty acids. C18:0 --> ~9 C18:1
• When the substrate is already unsaturated,
19.7.1 DESATURATION OF FATTY ACIDS IN subsequent double bonds are inserted
ANIMALS between the double bond nearest the car-
boxyl carbon, and the carboxyl carbon itself,
In animals, the substrates for desaturation are in such a way as to (usually) maintain the
derived from dietary fat or from the products methylene-interrupted distribution of dou-
of cytoplasmic fatty acid synthesis and elonga- ble bonds (Figure 19.7).
tion. The process of desaturation takes place
CH3(CH2)7CH = CH(CHz)7COSCoA
on the desaturase complex, which is located
~9-0IeoyICoA
on the endoplasmic reticulum and consists of
three components: NADH-cytochrome b s 1 ~6 desaturase
reductase; cytochrome b s; and the desaturase
CH3(CHZ)7CH =CHCHzCH =
enzyme. There is some doubt as to the true
CH(CH2)4COSCoA
number of desaturase enzymes. It is normally
~9.6-linoleoyl-CoA
assumed that there are four, which are desig-
nated ~9, ~6, ~s or ~4 fatty acyl-CoA desatu- • In a metabolic pathway leading to the for-
rases. The ~ notation indicates the position in mation of polyunsaturated fatty acids
Formation of unsaturated fatty acids 263
(PUFAs), desaturation usually alternates occurs entirely in the chloroplast, the formation
with elongation. of a-linolenic acid occurs from oleate-contain-
ing monogalactosyldiacylglycerol as the sub-
strate.
19.7.2 DESATURATION OF FATTY ACIDS IN
PLANTS
19.7.3 ESSENTIAL FATTY ACIDS
The desaturase complex in plants is less well
characterized. It appears to be similar to that in In mammals, PUFAs can be grouped into four
animals, with the exception that ferredoxin distinct families which differ in the number of
replaces cytochrome bs as the electron carrier. carbon atoms between the terminal methyl
Of particular importance is the fact that in group and the nearest double bond. This pat-
plant tissues, double bonds can be inserted tern arises because mammalian fatty acid
into a fatty acid between the methyl carbon desaturases cannot insert a double bond into a
and carbon 10 or a pre-existing double bond. fatty acid between carbon 10 and the methyl
Thus, plants can synthesize linoleic acid (a9,12 carbon, referred to in older texts as the (I)-car-
CI8:2) and a-linolenic acid (a9,12,IS CI8:3), bon. The families of PUFAs are derived from
whereas animals cannot. four precursor fatty acids. These fatty acids
Desaturation takes place both in the chloro- and the notation used to identify them are
plast stroma and on the endoplasmic reticulum. shown in Table 19.1.
The initial step in the de saturation process, PUFAs in each of the families are produced
which occurs in the chloroplast stroma, appears from their respective precursor fatty acid by a
to be elongation of newly synthesized palmi- series of alternating elongation and desatura-
toyl-ACP to give stearoyl-ACP. Stearoyl-ACP is tion reactions. A sequence of reactions for the
the substrate for a stearoyl-ACP desaturase n-9, n-6 and n-3 family of PUFAs is shown in
found in the chloroplast and is converted to Table 19.2. Note that for each of the fatty acids
oleoyl-ACP. Subsequent fatty acid desaturation within a family, the number of carbon atoms
can take place by two mechanisms, the eukary- between the methyl carbon and the nearest
otic pathway and the prokaryotic pathway, and double bond remains unchanged, regardless
appears to occur once fatty acids have been of subsequent elongations and de saturations.
incorporated into complex lipids (phospho- Until recently it was assumed that a4 dou-
lipids and glycolipids). In the eukaryotic path- ble bonds were inserted by a a4 desaturase.
way, so-called because it involves reactions in There is now evidence, at least in the case of
both the chloroplast and the endoplasmic retic- docosahexaenoic acid (DHA), that an alterna-
ulum, oleoyl-ACP is converted to oleoyl-CoA in tive mechanism operates. This involves the
the chloroplast and then transported to the elongation of eicosapentaenoic acid (EPA) by
endoplasmic reticulum. Here, it is incorporated four carbon units to produce a 9,12,IS,18,21 C24:5,
into phosphatidylcholine. In this phospholipid which is then acted upon by a6 desaturase to
form, the oleic acid is desaturated to linoleic give a 6,9,12,IS,18,21 C24:6. This fatty acid under-
acid by a12 desaturase. The linoleoylphos- goes chain shortening by ~-oxidation to pro-
phatidylcholine is transported back into the duce DHA. It remains to be established if this
chloroplast, where the phosphocholine moiety mechanism applies generally to the introduc-
is removed and the resulting diacylglycerol is tion of a4 double bonds.
converted to monogalactosyldiacylglycerol. At Since linoleic and a-linolenic acids cannot
this point linoleic acid is converted to a- be synthesized by mammals, they are termed
linolenic acid by a als desaturase. In the essential fatty acids (EFAs). PUFAs derived
prokaryotic pathway, so named because it from them are important as components of
Table 19.1 Precursor fatty acids of the four families of polyunsaturated fatty acids
Fatty acid Structure Family
Palmitoleic acid CHiCH2) 5CH=CH-(CHZ> 7COOH n-9 family

Oleic acid CH3(CHz> 7-CH= CH- (CH2) 7COOH n-7 family
Linoleic acid CH3(CHz> 4CH = CH-CH2-CH = CH-(CH2) 7COOH n-6 family
Linolenic acid CH3CH2CH = CH-CH2-CH = CH-CH2-CH = CH- (CHz> 7COOH n-3 family
membrane phospholipids and as precursors of substrate specificity of the !l6 desaturase is

prostaglandins, prostacydins, thromboxanes greatest for a-linolenic acid and lowest for oleic
and leukotrienes, which are important regula- acid, and therefore the relative rates of desatu-
tory compounds. ration of the precursor fatty acids are a-
Table 19.2 shows that the !l6 desaturase can linolenic > linoleic > oleic. When an animal is
introduce a double bond into each of the pre- fed a balanced diet, the intake of linoleic acid is
cursor fatty acids in the n-9, n-6 and n-3 fami- usually greater than that of a-linolenic acid,
lies. When all three precursors are present they which ensures an adequate supply of arachi-
compete for the same enzyme; however, the donic acid via the n-6 pathway. In cases of
Table 19.2 Metabolic transformations of the n-9, n-6 and n-3 fatty acid
families by desaturation and elongation
n-9 family n-6 family n-3 family

PRECURSOR £l9-C18:1 £l9,12_C18:2 £l9,12,15_C18:3
Oleic acid Linoleic acid a-Linolenic acid
Desaturation .1.6,9-C18:2 .1.6,9,12_C18:3 .1.6,9,,2,15_C18:4

.1.6 'Y-Linolenic acid
I I
Elongation .1.8,I1_C20:2 .1.8,11,14_C20:3 .1.8,11,14,17-C20:4
I I I I
Desaturation .1.5,8,I1-C20:3 .1.5,8,11,14_C20:4 .1.5,8,11,14,17-C20:5
.1.5 Arachidonic acid EPN
I I I
Elongation .1.7,10,13-C22:3 .1.7,10,13,16_C22:4 .1.7,10,13,16,19_C22:5
I I I I
Desaturation .1.4,7,10,13-C22:4 .1.4,7,IO,13,16_C22:5 .1. 4,7,IO,13,16,19_C22:6
.1.4 DHA*
Alternative mechanism for the synthesis of DHA
.1.7,1O,13,16,19_C22:5
EPA
Elongation I
.1.9,12,15,18,2I_C24:5
Desaturation I
.1.6 .1.6,9,12,15,18,21_C24:6
~-oxidation I -2C
.1.4,7,10,13,16,19_C22:6
DHA*
• EPA, Eicosapentaenoic acid; DHA, docosahexaenoic acid

Synthesis of triacylglycerols 265
essential fatty acid deficiency, the availability deficient. The ratio falls markedly within the
of oleic acid relative to linoleic acid is increased. first few days of birth as the polyunsaturated
The result is an enhanced de saturation of oleic fatty acids in milk are absorbed.
acid which leads to an increase in the tissue
content of the n-9 fatty acid, A5,8,1l_20:3, relative
19.8 SYNTHESIS OF TRIACYLGLYCEROLS
to the n-6 fatty acid, A5,8,1l,14_C20:4. Changes in
the ratio of these two fatty acids, sometimes Triacylglycerols are the main storage lipids in
referred to as the triene-tetraene ratio, are used animal and plant tissues. In animals, adipose
as an indicator of essential fatty acid deficiency. tissue depots, distributed widely around the
Until recently a ratio of greater than 0.4 has body and identified by anatomical location (e.g.
been taken as an indicator of an inadequate subcutaneous, perinephric and mesenteric), are
supply of essential fatty acids in humans; how- the major sites of triacylglycerol storage. In
ever, recent research has suggested that the some plants, triacylglycerols may be stored in
ratio should be lower, probably around 0.2. the seeds. This is particularly true of the seeds
EF A deficiency is best characterized in the of certain plants such as rape, sunflower, soya-
rat, where typical symptoms are markedly bean and linseed where the oil (triacylglycerol)
reduced growth, scaly paws and tail, reduced content can be as high as 400-500 g kg-I.
fertility, elevated respiratory quotient, high The structure of triacylglycerols is discussed
metabolic rate and high skin permeability to in Chapter 4. In animals, the fatty acids con-
water. The fatty acids necessary to prevent tained within triacylglycerols are derived
deficiency symptoms in the rat are linoleic acid either from dietary lipids following digestion,
(A9,12 C18:2) of plant origin, or arachidonic acid or by endogenous synthesis of fatty acids as
(A5,8,1l,14 C20:4) which can be formed in animal described earlier in this chapter. There are two
tissues from linoleic acid. Two other fatty acids major pathways for the synthesis of triacyl-
related to linoleic acid, in that they have a sim- glycerols. The first, the 2-monoacylglycerol
ilar methyl-terminal (n-6) chain structure, are pathway, is most active in the intestinal
also effective: A8,1l,14 C20:3 and A6,9,12 C18:3 ("{- mucosa and derives its substrates from lipid
linolenic acid). a-Linolenic acid (A9,12,15 C18:3), digestion (described in Chapter 28). The sec-
an n-3 fatty acid, is effective in promoting ond is the glycerol-3-phosphate pathway,
growth but does not prevent the skin deficien- which occurs in most tissues.
cy symptoms. Thus, fatty acids related struc-
turally (at the methyl end) to linoleic acid, the
19.8.1 THE 2-MONOACYLGLYCEROL PATHWAY
n-6 family, appear to be the true EFAs.
Grazing ruminants have a diet which is rich The main end products of dietary triacylglyc-
in linoleic acid (A9,12 C18:2) and a-linolenic acid erol digestion in monogastric animals are 2-
(A9,12,15 C18:3). However, as a result of the activ- monoacylglycerols and free fatty acids. These
ity of rumen microorganisms, most of these products are incorporated into mixed micelles
fatty acids are biohydrogenated to oleic and in the jejunum and ileum. The contents of the
stearic acids. Despite this, sufficient PUF As micelles are absorbed into the cells of the
escape biohydrogenation and pass to the small intestinal mucosa, and it is here that the resyn-
intestine where they are absorbed and utilized thesis of triacylglycerols takes place. The path-
very efficiently. Neonatal ruminants are born way is a simple two-step process which
with relatively low levels of essential fatty involves the sequential addition of two fatty
acids. The plasma triene-tetraene ratio in acids to 2-monoacylglycerol acceptor mole-
neonatal lambs, kids and calves immediately cules catalysed by monoacylglycerol acyl trans-
post-partum is considerably higher than 0.4, ferase (step 1 in Figure 19.7) and diacylglycerol
which suggests that ruminants are born EFA- acyltransferase (step 2 in Figure 19.7).
2-rnonoacylglycerol 1,2-diacylglycerol triacylglycerol

Figure 19.7 Synthesis of triacylglycerols by the 2-monoacylglycerol pathway.
The enzymes of this pathway are located 19.8.2 THE GLYCEROL-3-PHOSPHATE

on the smooth endoplasmic reticulum. PATHWAY
Monoacylglycerol acyltransferase is most
active on 2-monoacylglycerol substrates con- This is the major pathway of triacylglycerol
taining short-chain saturated or long-chain synthesis in tissues other than monogastric
unsaturated fatty acids. The enzymes for this intestinal mucosa. The glycerol backbone is
pathway are found in other tissues such as provided by glycerol-3-phosphate, most of
liver and adipose tissue; however, as the con- which is derived from glucose via dihydroxy-
centration of 2-monoacylglycerols is very low acetone phosphate (Figure 19.8).
in these tissues, their contribution to triacyl- Small amounts of glycerol-3-phosphate are
glycerol synthesis is negligible under normal synthesized by the direct phosphorylation of
circumstances. It is also important to remem- glycerol by the enzyme glycerol kinase
ber that this pathway is not normally impor- (Figure 19.9).
tant in ruminant intestinal mucosa because The production of triacylglycerols from
dietary triacylglycerols are hydrolysed to glycerol-3-phosphate is shown in Figure 19.10.
glycerol and free fatty acids in the rumen, and Initially a fatty acid is added to the 1 position
very little lipid escapes the rumen to be of glycerol-3-phosphate to produce I-acyl
digested in the small intestine. However, glycerol-3-phosphate. This reaction is catal-
when protected fat (triacylglycerols coated in ysed by glycerol-3-phosphate acyltransferase
formaldehyde-treated casein to prevent which has a preference for saturated fatty
hydrolysis of dietary lipid by rumen microor- acids. Addition of a fatty acid to the 2 position
ganisms) is fed to ruminants, large quantities is then catalysed by l-acylglycerol-3-phos-
of triacylglycerol are digested in the small phate acyltransferase, which has a preference
intestine and the 2-monoacylglycerol path- for unsaturated fatty acids. The product is a
way becomes more important. 1,2-diacylglycerol-3-phosphate, more com-
yH 2-OH
b HOCH
Glycerol-3-phosphafe I
dehydrogenase CH 2-O®
Dihydroxyacetone Glycerol-3-phosphate
phosphate
Figure 19.8 Production of glycerol-3-phosphate from dihydroxyacetone phosphate.

Synthesis of triacylglycerols 267
ATP ADP
reticulum. This dual distribution appears to be
.. \-./ a regulatory mechanism, as only the mem-

brane-bound enzyme is active. Factors which
Glycerol Kinase favour the synthesis of triacylglycerols, such as
energy intake in excess of requirements,
Glycerol Glycerol-3-phosphate increase the proportion of bound enzyme to
free enzyme. These effects are probably medi-
Figure 19.9 Production of glycerol-3-phosphate
from glycerol. ated via changes in hormonal status, e.g. an
increase in plasma insulin concentration.
monly known as phosphatidic acid.

19.8.3 TRIACYLGLYCEROL SYNTHESIS IN
Phosphatidate phosphohydrolase releases the
PLANTS
phosphate from the 3 position of phosphatidic
acid to produce a diacylglycerol, which is then In plants, triacylglycerol synthesis occurs via
further acylated by 1,2-diacylglycerol acyl- phosphatidic acid. Fatty acid synthesis in the
transferase to yield a triacylglycerol. plastid produces palmitoyl-ACP, which is
Phosphatidic acid is a common precursor of converted predominantly to oleoyl-ACP by
both triacylglycerols and phospholipids. The fatty acid elongation and desaturation.
amount of triacylglycerol formed from phos- Plastid fatty acyl-ACPs can be converted to
phatidic acid is determined by the activity of fatty acyl-CoAs and exported to the cyto-
the enzyme phosphatidate phosphohydro- plasm where they may be used for the syn-
lase. This enzyme can exist both free in the thesis of phosphatidic acid on the endoplas-
cytoplasm and bound on the endoplasmic mic reticulum. The pattern of fatty acid distri-
Glycerol-3-phosphate 1-acyt glycerol- 3-phosphate Phosphatidic acid
Pi
oII oII
o CH i O-C-R 1 o CHiO-C-R1
R2-C-O
-,
I 0
II
R2 -C-0-1
CHiO-C-R3 CHiOH
Triacylglycerol 1,2-diacylglycerol
Figure 19.10 Synthesis of triacylglycerols by the glycerol-3-phosphate pathway. Enzymes: 1, glycerol-3-

phosphate acyltransferase; 2, I-acyl glycerol-3-phosphate acyltransferase; 3, phosphatidate phosphohy-
drolase; 4, I,2-diacylglycerol acyltransferase.
bution in this phosphatidic acid is usually The second strategy involves the activation
palmitic acid in position 1 and oleic acid in of the polar head groups, choline or
position 2. Hydrolysis of the phosphate ethanolamine.
group from position 3 yields a diacylglycerol
Choline + ATP -> Phosphocholine + ADP
which is further acylated to triacylglycerol. In
Phosphocholine + CTP ->
many seeds the triacylglycerols are particu-
CDP-choline + PPi
larly rich in PUFAs (linoleic acid in soyabean,
sunflower seeds and safflower seeds; Diacylglycerol, produced by the dephos-
linolenic acid in linseed). This enrichment is phorylation of PA, displaces CMP from CDP-
apparently achieved by conversion of part of choline or CDP-ethanolamine to produce PC
the diacylglycerol pool to phosphatidyl- or PE, respectively. In animals, PS can be pro-
choline, which is subject to the action of duced from PE by head-group exchange,
desaturases on the endoplasmic reticulum. As whereas in plants PS synthesis occurs mainly
this conversion is reversible in many plants, by the CDP-diacylglycerol route plastids
the diacylglycerol pool becomes enriched (Figure 19.11). In animals, synthesis of phos-
with PUFAs leading to the production of pholipids occurs mainly on the smooth endo-
PUFA-enriched triacylglycerols. plasmic reticulum. However, in plants synthe-
sis of most phospholipids, but not PC and PI,
also occurs in chloroplasts and other non-pho-
19.9 PHOSPHOLIPIDS
tosynthetic organelles.
Phosphatidic acid is the basic building block
for the synthesis of a wide range of phospho-
19.10 GLYCOLIPID SYNTHESIS
lipids. Two strategies are employed to
achieve their synthesis. The first strategy The principal glycolipids of plant tissue are
leads to the production of phosphatidylserine monogalactosyldiacylglycerols (MGDGs) and
(PS), phosphatidylinositol (PI), phosphatidyl- digalactosyldiacylglycerols (DGDGs) with
glycerol (PG) and cardiolipin, and involves small amounts of tri- and tetragalactosyl deriv-
the synthesis of an activated diacylglycerol, atives. These lipids are almost exclusively con-
CDP-diacylglycerol. fined to chloroplast membranes. The synthesis
of MGDG requires the production of UDP-
Phosphatidic acid + CTP ->
galactose. Galactosyltransferases catalyse the
CDP-diacylglycerol + PPi
transfer of galactose firstly to diacylglycerol
PS, PI and PG are produced from CDP-dia- (DAG) to yield MGDG, and secondly to
cylglycerol by exchange of the CDP group MGDG to produce DGDG.
with serine, inositol or glycerol-3-phosphate,
DAG + UDP-Gal -> MGDG + UDP
respectively. Cardiolipin is produced by the
MGDG + UDP-Gal -> DGDG + UDP
condensation of two molecules of PG with the
elimination of glycerol (Figure 19.11). An alternative pathway for synthesis of
Phosphatidylethanolamine (PE) can be DGDG and the small quantities of tri- and
formed by the decarboxylation of PS and sub- tetragalactosyldiglycerides involves the trans-
sequently converted to phosphatidylcholine fer of a galactose residue from one molecule of
(PC) by three successive transmethylation MGDG to the galactose residue of another
steps in which the methyl groups are donated molecule of MGDG.
by S-adenosylmethionine (SAM). This route of
2 MGDG -> DGDG + DAG
PC synthesis is of only minor importance in
plant and animal tissues, but it is the main Analysis of the positional distribution of
pathway of PC synthesis in bacteria. fatty acids in monogalactosyldiacylglycerols
Glycolipid synthesis 269
PA
CTP
Pi PPi
DAG CDP-DAG G-3-P
G
Eth
~PG
CDP-Cho CDP-Eth
~
Ser Inos
CMP CMP CMP glycerol
PC PE PS PI CL
~~
3 x SAHC 3 x SAM CO 2
Figure 19.11 Summary of the synthesis of phospholipids. PA, phosphatidic acid; pc, phosphatidyl-
choline; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phos-
phatidylglycerol; DAC, diacylglycerol; crp, cytidine triphosphate; CDP, cytidine diphosphate; SAM, S-
adenosyl methionine; SAHC, S-adenosyl homocysteine; CDP-Cho, CDP-choline; CDP-Eth,
CDP-ethanolamine; Eth, ethanolamine; Ser, serine; CL, cardiolipin; Inos, inositol.
from a wide variety of plants has revealed that at position 2, or C18:1 at position 1 and C16:3
there are two distinct groupings of plants. One at position 2.
group, typified by the bryophytes, pterido- In contrast, synthesis of the C18:3 galac-
phytes, gymnosperms, and some angiosperms tolipids involves both the endoplasmic reticu-
of the spinach, potato and bras sica families, lum and the chloroplast. It utilizes the pathway
contains an unusual fatty acid, hexadeca- of phosphatidylcholine synthesis. On the
trienoic acid (C16:3), at the 2 position of the endoplasmic reticulum, DAG containing C18:1
MGDG. These plants are referred to as C16:3 in positions 1 and 2 is converted to phos-
galactolipid plants. In more advanced phatidylcholine, and the fatty acids undergo
angiosperms such as the Fabaceae, Asteraceae desaturation to linoleic acid (C18:2). The phos-
and Poaceae, C16:3 is virtually absent and is phatidylcholine is then transferred to the
replaced by linolenic acid, C18:3. These plants chloroplast where the phosphocholine residue
are referred to as C18:3 galactolipid plants. is removed to yield a l-linoleyl-2-linoleyl dia-
Synthesis of C16:3 galactolipids takes place cylglycerol from which MGDG is synthesized.
entirely in the chloroplasts. This pathway The MGDG fatty acids are desaturated to C18:3
results in the initial production of diacylglyc- prior to transfer of a second galactose residue
erols with C16:0 at the 2 position and C18:1 in to produce DGDG.
the 1 position. Following addition of galactose Interestingly, the sulphur-containing gly-
at the 3 position to produce MGDG, the fatty colipid of chloroplasts, sulphoquinovosyl dia-
acids undergo de saturation to produce MGDG cylglycerol, when synthesized entirely in the
containing either C18:3 at position 1 and C16:0 chloroplast (the prokaryotic pathway), con-
tains very little C16:3 in the 2 position and the conversion of sphinganine to sphingosine
usually has C18:3 in the 1 position. When by the removal of two hydrogens. This pro-
synthesized from the 1-linoleyl,-2-linoleyl duces the simplest type of sphingolipid,
diacylglycerol pool originating on the endo- referred to as a ceramide (Figure 19.12).
plasmic reticulum (the eukaryotic pathway), Cerami des can undergo transformation to
it contains two C18:3 groups. the four main types of sphingolipids:
• sphingomyelins
19.11 SYNTHESIS OF SPHINGOLIPIDS • cerebrosides (also called monoglycosyl
ceramides)
Sphingolipids are found in both plants and
• neutral glycosphingolipids (polyglycosylat-
animals, but are quantitatively more important
ed ceramides)
in the latter. The synthesis of sphingolipids
• gangliosides (polyglycosylated ceramides
starts with the condensation of palmitoyl-CoA
containing N-acetylneuraminic acid).
with serine to produce 3-ketosphinganine,
which undergoes hydrogenation at the 3-keto The conversion to sphingomyelins is
group to yield sphinganine (also referred to as brought about by the transfer of phospho-
dihydrosphingosine ). choline from phosphatidykholine to the free
This is followed by the addition of a fatty hydroxyl group on carbon 1 of sphingosine.
acid via the amino group of sphinganine and Cerebrosides are formed by the transfer of a
o
II
CH3(CH2)14COSCoA + NH2
-CHCOOH
I
--------+. CH3(CH~14C-CHCH20H
I
CH 20H NH2
3-ketosphinganine
Palmitoyl-CoA Serine
NADPH+H+~
NADP++-1
OH OH
I
CH3(CH2)14C1:CH20H 44-7--::---,::::---- I
CH3(CH2)14CHCHCH20H
I
NH2
C::()
I
CoASH fatty acyI-CoA Sphinganine
R
N-acyl sphinganine
Ceramide
(N-acyI sphingosine)
Figure 19.12 Synthesis of cerarnides.

Biosynthesis of terpenes and sterols 271
sugar residue to the hydroxyl on carbon 1 of 19.12 BIOSYNTHESIS OF TERPENES AND
sphingosine from a UDP-sugar. The most com- STEROLS
mon sugars found in cerebrosides are galactose Squalene is a linear triterpene from which
and glucose. Neutral glycosphingolipids are plant and animal sterols are derived. The vari-
synthesized from cerebrosides and may contain ous stages of its synthesis, which occurs in the
six or more sugar units, mainly galactose, glu- cytoplasm, involve a number of terpenoid
cose or N-acetylgalactosamine. Gangliosides intermediates and are typical of the reactions
are the most complex form of sphingolipids: undergone during the synthesis of many of
they are characterized by the presence of N- the higher terpenoids. This section outlines
acetylneuraminic acid (sialic acid) as the termi- the reactions involved in the production of
nal sugar unit (Figure 19.13). squalene from acetate and its subsequent con-
It is now clear that sphingolipids are
version to plant and animal sterols.
important in the process of cell recognition.
The specific sphingolipid composition of the
19.12.1 SYNTHESIS OF MEVALONIC ACID
plasma membrane contributes to the cell's
chemical fingerprint, and changes in mem- Experiments have shown that all the carbon
brane sphingolipid composition during tissue atoms in the carbon skeleton of squalene are
growth are thought to be related to regula- derived from acetate. The initial stages of
tion of cell-to-cell contacts. It has been shown squalene synthesis involve the condensation
that sphingolipid composition of blood cells of two molecules of acetyl-CoA to yield ace-
is one of the determinants of blood group. toacetyl-CoA, followed by the addition of a
Genetic defects in sphingolipid metabolism third molecule of acetyl-CoA to give hydroxy-
can have fatal consequences. Two genetically methylglutaryl-CoA (HMG-CoA) (Figure
inherited disorders of sphingolipid metabo- 19.14).
lism, Tay-Sachs disease and Niemann-Pick The synthesis of HMG-CoA is also the first
disease, result in early infant death. stage in the synthesis of ketone bodies from
PC DAG
Ceramide Sphingomyelin
UDP-Gal UDP
Ceramide Cerebroside
(UDP-sugar)n (UDP)n
Cerebroside V. Neutral glycoaphingolipids
(UDP-sugar)n (UDP)n
(CMP-NANA)n (CMP)n
Cerebroside V. Gangliosides
Figure 19.13 Conversion of ceramides to sphingomyelin, cerebrosides, neutral glycosphingolipids and

gangliosides.
CH 3
I
C=O
I COOH
CH3 S I
I
CH3 C=O JoA CH 2
I
2 X C=O
I
l I
HO-C-CH3
o
CH2
----~-~-----.~
®
I I I
S C=O ~H2
I I
CoA C=O
S I
I
CoA S
Acetyl-CoA I
CoA
Acetoacetyl-CoA
(HMG-CoA)
Figure 19.14 Synthesis of hydroxymethylglutaryl-CoA (HMG-CoA).
acetate in liver mitochondria (Chapter 13). and is catalysed by the enzyme HMG-CoA
HMG-CoA synthetase catalyses step 2 in both reductase, which is thought to be rate-limiting
processes, and is therefore found in both the in cholesterol synthesis in animals (Figure
mitochondria and the cytoplasm. However, 19.15).
the mitochondrial and cytoplasmic synth- It has been shown that cholesterol synthesis
etases differ in their catalytic properties and is suppressed by increased consumption of
isoelectric points. cholesterol. There is evidence, at least in the
From this point the pathways of ketone rat, that HMG-CoA reductase shows changes
body and squalene synthesis diverge. In the in activity related to the pattern of food con-
synthesis of ketone bodies, HMG-CoA is sumption. This is thought to be a response to
cleaved to yield acetoacetate and acetyl-CoA. the loss of free cholesterol and its derivatives
The acetoacetyl-CoA may then be metabolized such as bile acids. The enzyme has a relatively
to l3-hydroxybutyryl-CoA. In the synthesis of short half-life (2-4 h) and alterations in its
squalene, HMG-CoA undergoes conversion to activity are due mainly to changes in its rate of
mevalonic acid, a reaction in which the CoA synthesis. In addition, HMG-CoA reductase
group is lost, and the resulting carboxyl group activity is also regulated by covalent modifica-
is reduced to an alcohol using NADPH as a tion via a phosphorylation/dephosphorylation
reducing agent. This reaction is irreversible mechanism.
COOH
I
CH 2
I
HO-C-CH3
I
CH 2
I
c=o HMG-CoA RiJductase
I
S
I
CoA
HMG-CoA Mevalonic acid
Figure 19.15 Synthesis of mevalonic acid.
19.12.2 CONVERSION OF MEVALONIC ACID phosphomevalonate kinase and also requires
TO SQUALENE ATP. Another molecule of ATP is consumed in
a complex reaction catalysed by pyrophos-
Mevalonic acid is first phosphorylated to pro- phomevalonate decarboxylase, in which
duce mevalonate-5-phosphate, a reaction mevalonate-5-pyrophosphate is converted to
requiring ATP and catalysed by mevalonate isopentenyl pyrophosphate. Some isopen-
kinase. This is followed by the addition of a tenyl pyrophosphate undergoes isomeriza-
second phosphate group to yield mevalonate- tion to form dimethylallyl pyrophosphate
5-pyrophosphate. This reaction is catalysed by (Figure 19.16).
Meva/onate kinase
7,\
ATP ADP
Mevalonic acid Mevalonate-5-phosphate
ATP ~
Phosphomevatonate
kinase
ADP
Pyrophosphomeva/onate
decarboxytase
lsopentenyl pyrophosphate Mevalonate-5-pyrophosphate

(IPP)
1l p;;;~;:r;,r:te
Isomerase
CH 3
I
C-CH 3
II
CH
I
CH 20 - ® 0
Dimethylallyl pyrophosphate
(DMAPP)
Figure 19.16 Conversion of mevalonic acid to isopentenyl pyrophosphate and dimethylallyl pyrophos-
phate.
The synthesis of squalene continues with are examples of 'head-to-tail' condensations,
the condensation of isopentenyl pyrophos- the nomenclature referring to the methylene
phate (IPP) and dimethylallyl pyrophosphate or methyl end of IPP and DMAPP as the 'tail'
(DMAPP) to form geranyl pyrophosphate. end and the pyrophosphate end as the 'head'
This in turn condenses with another molecule end (Figure 19.17).
of IPP to yield farnesyl pyrophosphate. The Squalene is synthesized by the subsequent
enzyme responsible for both of these conden- 'head-to-head' condensation of two molecules
sations is prenyl transferase. These reactions of farnesyl pyrophosphate. This reaction occurs
CH2 CH 3
II I
C-CH 3 C-CH 3
I + II
CH 2 CH
0-®-®
I I
CH 2 CH20-®-®
lsopentenyl pyrophosphate Dimethylallyl pyrophosphate

(IPP) (DMAPP)
~PP;
,,
,
,,
I
,,
I
J'nen)dtr.sns~r.sse
\
\
\
\
\
\
\
\
\ Geranyl pyrophosphate
\
\
\
\
~
IPP
PPi
Famesyl pyrophosphate
Figure 19.17 Synthesis of farnesyl pyrophosphate.
CH 3 CH 3 CH 3
I I I
@-®-o-CH;CH=C-CHPHicH=C-CHPHi"CH=C-CH3
FarnesylpY"3Phosphate
NADPH+ H+
NADP+ + 2PPi
CH 3 CH 3 CH 3
I I I
CH;-C=CH-CHPH~=CH-CH;CH~=CH-CH;CH;CH'-CHPHPH=y-CHPH;CH=<r-CH3
CH3 CH 3 CH 3
Squalene
Figure 19.18 Condensation of two molecules of farnesyl pyrophosphate to produce squalene. Inset: alter-
native structure of squalene showing its similarity to the steroid nucleus.
in two steps, catalysed by the presqualene syn- cyclization to yield lanosterol or cycloartenol is
thase and squalene synthase (Figure 19.18). catalysed by cyclase enzymes. Cycloartenol is
Squalene then undergoes cyclization to form further metabolized to stigmasterol, and lanes-
lanosterol in animals and fungi, or cycloartenol terol to ergosterol and cholesterol (Figure
in plants and algae. The process involves a two- 19.19). The detail of these transformations is still
step reaction in which squalene is first converted unknown, but that of cholesterol synthesis is
to squalene-2,3-epoxide in a reaction catalysed thought to involve about 20 reactions located
by squalene monooxygenase. Subsequent on the endoplasmic reticulum.
Squalene-2,3-epoxide
HO HO
Lanosterol Cycloartenol
HO HO
Cholesterol Stigmasterol
Figure 19.19 Cyc1ization of squalene to form sterols.

In addition to its function in cell mem- In plants, geranyl pyrophosphate, farnesyl
branes, cholesterol is the precursor of a num- pyrophosphate and geranylgeranyl pyrophos-
ber of important compounds, including phate (produced by the condensation of farne-
cholecalciferol (vitamin OJ which is involved syl pyrophosphate with isopentenyl pyrophos-
in the regulation of calcium metabolism and phate) are branch points for the synthesis of a
bone growth; the bile acids, essential for effi- number of non-sterol terpenes. For example the
cient digestion and absorption of dietary latter provides the carbon backbone for the syn-
lipids; and the steroid hormones, which reg- thesis of the gibberellins, the carotenoids, the
ulate many aspects of growth, development tocopherols and tocotrienols, vitamins KJ and
and metabolism. K:z and the phytol side chain of chlorophyll.
SYNTHESIS OF AMINO ACIDS 20
20.2 Assimilation of nitrate 279
20.3 Nitrogen fixation 281
20.3.1 Molecular biology of nitrogen fixation 282
20.4 Assimilation of ammonia 282
20.5 Biosynthesis of amino acids 284
20.5.1 Aromatic amino acids and related 284
compounds
20.5.2 Branched-chain aliphatic amino acids 285
20.6 Nutritional role of amino acids 287
20.7 Herbicides and amino acid biosynthesis 289
20.1 INTRODUCTION The main sources of nitrogen for non-legu-

Plants and microorganisms can synthesize minous plants are NO; or NHr from the soil.
These are mainly derived from fixation of
amino acids from simple nitrogen com-
pounds such as nitrate or ammonia, or from atmospheric nitrogen gas by chemical means
or by free-living bacteria and blue-green
those released during protein turnover.
Animals cannot form amino acids from inor- algae in the soil, or by these organisms in
symbiotic associations with higher plants. In
ganic nitrogen compounds, but obtain them
most soils, nitrate is more abundant than
from protein in the diet or from the break-
ammonia because NHr is readily oxidized to
down of body proteins.
NO.1 by nitrifying bacteria. In some grassland
In addition to the 20 amino acids found in
proteins, some plants have pathways which and forest soils, where nitrification is inhibit-
enable them to make other, non-protein ed by low pH or phenolic compounds in the
amino acids. Some of these are toxic or are of soil, NHr may be the major form. Both can be
used by the plant but each probably inter-
commercial significance (Chapter 5).
feres with the utilization of the other, thus
Nitrogen is available to plants mainly as
nitrate (NO}), ammonia as the ammonium ion the presence of NHr may inhibit uptake and
(NHr) and nitrogen gas (N 2). metabolism of NO;.
NO; and NHr are readily available to plants
from the soil, but although 78% of the atmos- 20.2 ASSIMILATION OF NITRATE
phere consists of nitrogen gas, it is available
Any nitrate taken up by plants from the soil
only after reduction by prokaryotic microor-
must be converted into ammonia before it can
ganisms, either free-living or in symbiotic
be used to make organic nitrogen-containing
association in root nodules.
materials. This process takes place in two
Organic nitrogen-containing compounds
stages:
are rarely available and are not much used by
green plants. However, fungi are more depen- • conversion of nitrate (NO;) to nitrite (N0 2)
dent on them, and may be able to supply catalysed by the enzyme nitrate reductase;
nutrients to higher plants through their myc- • conversion of nitrite to ammonia (NHr)
orrhizal symbiotic associations with roots. catalysed by the enzyme nitrite reductase.
280 Synthesis of amino acids
Reduction of NO;- and N0 2 may take place N0 2 is very toxic to plants but, because the
predominantly in the roots or shoots, depend- activity of nitrite reductase is normally higher
ing on the species and the availability of nitro- than that of nitrate reductase, nitrite normally
gen (see Chapter 26). does not accumulate because it is rapidly con-
Nitrate reductase is a complex enzyme verted into NH;.
which is located in the cytoplasm. It contains N0 2 formed by the action of nitrate reduc-
FAD, haem and molybdenum. The conversion tase moves into the chloroplasts in the leaf or
of nitrate to nitrite requires a supply of a the proplastids in the roots, and is reduced in
reducing agent, which is usually NADH but these organelles by nitrite reductase. In chloro-
may sometimes be NADPH. The reaction takes plasts, reduced ferredoxin (FdH 2) formed by
place as shown in Figure 20.1a. light-driven electron transport is used as
Molybdenum forms an essential part of the reducing agent. In roots, it appears that
enzyme. Plants deficient in molybdenum can- NADPH, formed by oxidation of sugars by the
not utilize NO;- even if it is present in the soil. pentose phosphate pathway, may be used
The heavy metal tungsten can be incorporat- (Figure 20.1b).
ed into the enzyme in place of molybdenum, Both nitrate and nitrite reductase are
but the enzyme formed in this way is inactive. inducible enzymes. Although such enzymes
NADH FAD Cyt b Mo V N02'
WD> XFADH,X :: x MO"X NO,
b
NADPH + H+ NH/
or FdH2
Siroheme
NADP++ Fe"
or Fd
Figure 20.1 Mechanism of (a) nitrate reductase and (b) nitrite reductase. Nitrate reductase normally uses
NADH as a reducing agent, but nitrite reductase in different tissues may use NADPH or reduced ferre-
doxin.
Nitrogen fixation 281
are common in prokaryotic organisms they nitrogen to the enzyme and is an essential part
are unusual in eukaryotes. The activity of of the mechanism. The reaction requires a
nitrate reductase often limits the rate of pro- large input of energy and a reducing agent.
tein synthesis. Activity of this enzyme is con- Nitrogenase is a large and complex enzyme
trolled by regulation of the relative rates of its consisting of two types of protein.
synthesis and degradation. Light and high
• A small protein containing iron and consist-
concentrations of NO; in the cytoplasm both
ing of two subunits, each of molecular
increase activity of the enzyme, whereas its
weight about 30 kDa. This dimer contains
activity is decreased by NH;. The activity of
four atoms of iron associated with four sul-
nitrite reductase is also increased under these
phur atoms. The protein binds ATP.
circumstances.
• A large protein containing 28 iron and two
molybdenum atoms and consisting of 2a
20.3 NITROGEN FIXATION and 213 subunits. a subunits have a molecu-
lar weight about 56 kDa and 13 subunits
Vast quantities of nitrogen are present in the
about 60 kDa.
atmosphere in the form of N z but this molecule
is extremely stable and its nitrogen atoms are The Fe protein transfers electrons from the
available to living organisms only after being electron donor (reducing agent) to the Fe-Mo
reduced to NH;. This reduction is brought protein which actually reduces the nitrogen.
about through the process of nitrogen fixation, The reducing agent is reduced ferredoxin or
carried out either by prokaryotic organisms or flavodoxin. These compounds are converted
through the industrial synthesis of fertilizers. to their reduced forms by NADH or NADPH
Fixation of nitrogen cannot be achieved by produced by metabolism of leaf-derived sug-
eukaryotes functioning alone, although some ars in the nodules (Figure 20.2).
higher plants obtain reduced nitrogen com- Both the Fe-protein and the Fe-Mo protein
pounds by entering into symbiotic associations are denatured by oxygen, and nitrogenase
with prokaryotic nitrogen-fixing organisms by must therefore be protected from oxygen in
forming root nodules. Agriculturally the the root nodule. This is achieved through the
legumes are by far the most important class of anatomy of the nodule, and by the presence
plants of this type, but nitrogen fixation also in the nodules of the protein leghaemoglobin.
occurs in a number of pioneer species of trees This protein has a high affinity for oxygen
and shrubs over a wide range of genera. and is able to make oxygen available for respi-
Nitrogen is also fixed by soil- and water-living ration whilst preventing it from reaching the
bacteria, which make a considerable contribu- nitrogenase.
tion to the nutrition of their environments Nitrogen fixation in legumes requires very
from which other organisms benefit. close integration of the activities of the plant
Nitrogen-fixing prokaryotes, whether pre- and the bacteria. Thus the reducing agents
sent in root nodules or free-living, reduce needed for nitrogenase are derived from sug-
nitrogen to ammonia through action of the ars formed in the leaves and transported in the
enzyme nitrogenase which catalyses the fol- phloem. In addition, removal and assimilation
lowing reaction: of the ammonia into organic nitrogen com-
pounds also requires plant-supplied sugars.
N z + 16ATP + 8 electrons + 8H+ -->
Many of the components of the nodule, such
2NH3 + 16ADP + 16Pj + Hz Equation 20.1
as leghaemoglobin, are also products of plant
Production of hydrogen by reduction of genes. On the other hand, nitrogenase is
protons appears to be required for binding of encoded in bacterial genes.
Ferredoxin reduced
oxidised
(reduced)
Fe protein
Ferredoxin reduced oxidised

(oxidised)
Figure 20.2 Mechanism of nitrogenase. Electrons are transfered from reduced ferredoxin or flavodoxin to
an iron and then to an iron/molybdenum protein before reducing nitrogen gas.
20.3.1 MOLECULAR BIOLOGY OF NITROGEN ule-specific gene products or nodulins, which

FIXATION are involved in nitrogen and carbon metabo-
lism, nodule structure and regulation of oxy-
There is a great deal of interest in the molecu-
gen concentrations (leghaemoglobin).
lar biology of nitrogen fixation because of the
In addition to ammonia, nitrogenase also
possibility of increasing the efficiency of the
produces Hz as an essential part of its mecha-
process in legumes and of incorporating genes
nism of action (Equation 20.1). In some species
for nitrogen fixation into non-legumes. This
of Rhizobium this gas leaks into the soil and the
would have the major benefit of reducing fer-
energy associated with it is lost, reducing the
tilizer requirements. Although the process is
efficiency of the process. However in most
extremely complex, progress has been made in
Rhizobium species the Hz is oxidized to HzO by
understanding the means of regulation.
an uptake of hydrogenase which produces
In legumes, fixation takes place in the root
ATP in the process. Increasing the efficiency of
nodules, which are formed when soil-living
Rhizobium bacteria invade the root via root the hydrogenase may thus decrease the ener-
gy demands of nitrogen fixation.
hairs. A particular Rhizobium species generally
infects only one species of legume, and this
specificity is controlled by bacterial nod genes,
20.4 ASSIMILATION OF AMMONIA
which are required for successful nodulation.
A series of flavones, flavanones and isofla- Ammonia is very toxic to most cells as it
vanones, produced by different legumes, acti- appears to uncouple electron transport from
vate bacterial nod genes and form the basis of ATP production in mitochondria and chloro-
the specificity of the nodulation process. plasts. Therefore, however it is produced, it
Bacterial products cause curling of root hairs must be rapidly converted into non-toxic
and the start of the infection process. organic compounds. In green plants ammonia
Bacterial nif genes code for proteins respon- is produced by nitrogen fixation, nitrite reduc-
sible for the nitrogen fixation process itself. tion, uptake from the soil and as a result of the
Thus, nifH encodes the Fe protein, nifD the a operation of photorespiration (Chapter 17). It
subunit, and nifK the ~ subunit of the Fe-Mo has been estimated that the quantities of
component of nitrogenase. Other nif genes ammonia produced from photo respiration
control synthesis and processing of cofactors may be as much as 20-fold greater than those
and electron donors. Another series of bacteri- produced by uptake of nitrogen from the soil.
al genes, the fix genes, may be associated with The assimilation of ammonia is brought
ferredoxin production. about in a somewhat indirect way through the
Many plant genes are also required for action of two enzymes called glutamine syn-
effective nodulation. These include the nod- thetase (GS) and glutamate synthase
Assimilation of ammonia 283
(GOGAT). The reactions are shown in Figure In chloroplasts, GOGAT uses reduced ferre-
20.3. doxin produced during the light reactions as
C0 2 H
I
CH 2
I
CH 2
I
H-C- NH3
+
I
C0 2H
Glutamate
Glutamine
synthetase (GS)
CONH 2 C0 2H
I I
CH 2 CH 2
I I
CH 2 CH 2
I
H-C- NH3
+ I
H-C=O
I I
C0 2H C0 2H
Glutamine a-ketoglutarate
Glutamate
synthase (GOGA 1)
Fdor
NADP+
C0 2H
I
CH 2
I
CH2
I +
H-C- NH3
I
C02H
Glutamate
Figure 20.3 Reactions responsible for assimilation of ammonia by glutamine synthetase and glutamate
synthase in plants.
the reducing agent, but in non-green tissues energy. The pathways by which the amino
NADPH (or NADH) probably replaces FdHz as acids are synthesized are summarized in
the reducing agent. Figure 20.4.
Ammonia can also be converted to gluta-
mate by rumen microorganisms. This appears
20.5.1 AROMATIC AMINO ACIDS AND
to take place by amination of a-ketoglutarate
RELATED COMPOUNDS
by ammonia. The process allows ammonia
and other non-protein nitrogen sources to be Aromatic amino acids and other aromatic
used in the synthesis of microbial protein. compounds are synthesized by the shikimic
Glutamate may be further transformed into acid pathway from phosphoenolpyruvate and
glutamine, asparagine and ureides. These erythrose-4-phosphate. The outline of the
compounds, together with glutamate, serve as pathway is shown in Figure 20.5.
the main forms in which nitrogen is transport- Phosphoenolpyruvate (from glycolysis) and
ed around most plants. The transport of nitro- erythrose-4-phosphate (from the Calvin cycle
gen in plants is discussed in more detail in or pentose phosphate pathway) react together
Chapter 26. to form the seven-carbon compound, 3-deoxy-
o-arabinoheptulosonic acid-7-phosphate which
cyclizes to dehydroquinic acid. Loss of water
20.5 BIOSYNTHESIS OF AMINO ACIDS
and reduction lead to shikimic acid. A second
Although the formation of organic nitrogen molecule of phosphoenolpyruvate reacts with
compounds from NO}, NH; or N z takes place 5-phosphoshikimic acid and serves as the
only in plants and prokaryotes, pathways for source of the side-chain carbon atoms of the
the formation of amino acids from glutamate aromatic amino acids, resulting in production
occur in all organisms, although some cannot of chorismic acid. The aromatic amino acids are
synthesize all amino acids. synthesized from this compound.
In general, amino acids are all synthesized The pathway illustrates many of the gener-
in a similar way, by processes which are al features of pathways by which amino acids
almost the reverse of the steps which lead to are synthesized:
amino acid breakdown. Thus the carbon
• transamination of a-ketoacids gives rise to
skeletons are constructed first from com-
amino acids as one of the last steps;
pounds such as pyruvate, TCA cycle interme-
• ATP and NADPH are required;
diates or sugar phosphates, giving rise to a
• the starting materials are small compounds
series of a-ketoacids, one corresponding to
derived from the pathways of intermediary
each amino acid. These are then transaminat-
metabolism.
ed to form the a-amino acids. The amino
group donor is usually glutamate which is It is a relatively long pathway that is partic-
converted into a-ketoglutarate and which is ularly important in plants, which contain
therefore a key compound in amino acid many phenolic compounds. The pathway is
metabolism. absent from animals. Phenylalanine and tyro-
The pathways used in the synthesis of the sine or intermediates in the pathway act as
carbon skeletons of amino acids are usually precursors of other phenolic compounds.
branched so that members of an amino acid A particularly important reaction in plants
family may be formed from common interme- is the deamination of phenylalanine to cin-
diates. Some of the pathways involve only a namic acid (Figure 20.6), catalysed by the
few steps, but others may require 20 or more enzyme phenylalanine ammonia lyase (PAL).
reactions. These reactions, being biosynthetic The activity of this enzyme is increased by
in nature, are usually reductions and require light, and leads to increased production of
Biosynthesis of amino acids 285
(glucose]
o
.'""'T"OO.'' '. - ! ::"'"1
tryptophan . . - - 3-phosphoglycerate - glycine
phen.ylalanine ' - . . 11 cysteine
tyroSine .............. ~
PhOSPhOrOIPyrUvate
alanine
valine
pyruvate ---. leucine
~~~oA
~
.-/(
oxaloacetate
aspartate
asparagine
methionine
threonine
lysine
isoleucine
CL)-ketOgl~._______
I \
glutamate
glutamine
'" ;uc1cinYI-coA proline
" - - - - ." arginine
~
porphyrins
haem
chlorophyll
Figure 20.4 An outline of the pathways for the production of carbon skeletons required for amino acid
synthesis. The starting materials are mostly intermediates of glycolysis or the TCA cycle.
pigments, lignin and other phenolic com- branched pathway which is shown in Figure
pounds. Anthocyanins contain three rings as 20.7.
shown in Figure 5.8. The carbon atoms of the The synthesis of valine begins with conden-
B ring and central ring are formed from the sation of two molecules of pyruvate to form
shikimic acid pathway, and those of the A the five-carbon compound a-acetolactate. This
ring come from three molecules of acetyl- is reduced, dehydrated and transaminated to
CoA. form valine. Leucine arises from a-ketoiso-
valerate by a branch of this pathway. The syn-
thesis of isoleucine is similar to that of valine,
20.5.2 BRANCHED-CHAIN ALIPHATIC AMINO
but begins by condensation of pyruvate with a
ACIDS
molecule of a-ketobutyrate which contains an
The aliphatic amino acids, valine, leucine and extra carbon atom. This then undergoes a
isoleucine, are synthesized from pyruvate by a series of transformations which are similar to
COOH
CHO HO-t-
COOH
I
I
H-C-OH 6H 2
C-o..® I
H-C-OH bHOH
bH2 I bHOH
CH2~
I
-- --
Phosphoenolpyruvate Erythrose-4- H-C--
phosphate
~H2~
3-deoxy-D-arabino-
COOH heptulosonic acid-7-
phosphate
- -COOH-e;-o@)
I
CH 2 COOH
Phosphoenol-pyruvate
OH OH ---. ~
EPSP ---.
OH synthase CH 2
Shikimic acid O-lcoOH
OH
o Chorismic acid
6
CHz-lcOOH
Phenyl pyruvate OH
I
4-hydroxyphenylpyruvate
1
NH2
I
Hz-CH-COOH
NH2
~CH,..dH-COOH
~N)I
H
OH
Phenylalanine
Tyrosine Tryptophan
Figure 20.5 Pathway for biosynthesis of aromatic amino acids.The enzyme EPSP synthase is inhibited by
the herbicide glyphosate.
Nutritional role of amino acids 287
6
I:
NH2
I
6
CH-CH-COOH
CHrCH_COO:henytatanine ammonia
\
o/Bse
Phenylalanine trans-cinnamate
r02+ NADPH
l'-.NADP+ + H20
CH-CH-COOH
flavonoids lignin other

anthocyanins phenolics
Figure 20.6 The reaction catalysed by the enzyme phenylalanine ammonia lyase (PAL). This is the first
step in the pathway for the conversion of phenylalanine into a wide range of phenolic compounds in
plants.
those in the formation of valine. Steps in the use glutamate to make all of the other amino
synthesis of isoleucine and corresponding acids. Higher animals cannot make amino acids
steps in the synthesis of valine are catalysed by
from inorganic nitrogen compounds and thus
the same enzymes. require amino acids (usually in the form of pro-
tein) in their diets. They can make about half of
the 20 amino acids from other amino acids, but
20.6 NUTRITIONAL ROLE OF AMINO ACIDS
cannot make the remainder because they lack
Organisms differ in their ability to synthesize the enzymes required for their synthesis. These
amino acids. Bacteria and plants have the must therefore be supplied in the diet and are
enzymes needed to make glutamate from inor- termed essential amino acids (Table 20.1). Often
ganic nitrogen compounds such as NH3 or NO} these are the amino acids which have the most
and a source of carbon. They can also normally complex biosynthetic pathways.
Pyruvate
y
Acetohydroxy acid
synthase
Pyruvate
Acetohydroxy acid
synthase
a-ketobutyrate
CH 3
CH 3
~O d-o
I CH 3CH 2-LoH
CH 3-C-OH
booH
~OOH
a-aceto-a-
a-acetolactate hydroxybutyrate
1 CH 3
1
CH 3 I
CH 2
~H-CH3 ~H-CH3
Lo 6-0
/- bOOH
a-keto isovalerate
booH
a-keto-f3-methyl
valerate
~~ine j
j
CH 3
CH 3 I
CH 2
~H-CH3
~H-CH3
~HNH2
dHNH2
booH
booH
L-valine
L-isoleucine
Figure 20.7 The pathways for synthesis of aliphatic amino acids. The enzyme acetohydroxy acid synthase
is the site of action of the sulphonylurea herbicides.
A great deal of amino acid interconversion acids to the requirements of protein synthesis.
takes place to match the availability of amino This involves:
Herbicides and amino acid biosynthesis 289
Table 20.1 Essential and non-essen- rumen large numbers of microorganisms.
tial amino acids in the diet of non- These are able to make the whole range of
ruminant animals amino acids from nitrogen compounds in the
Essential Non-essential diet. These are then incorporated into the pro-
teins of the microorganism. These amino acids
Arginine Alanine are released, and become available to the ani-
Histidine Glycine mal as the microorganisms are degraded on
Isoleucine Aspartic acid passing through the digestive system. Thus
Valine Asparagine there is no dietary requirement for essential
Threonine Glutamic acid
Methionine Glutamine
amino acids for ruminants - provided that
Phenylalanine Proline they receive an adequate supply of nitrogen-
Tryptophan Hydroxyproline containing compounds in the diet they can
Leucine Serine obtain all of the amino acids from the microor-
Lysine Cysteine ganisms. Even so, under certain conditions
Cystine such as high levels of milk production or rapid
Tyrosine
growth, certain amino acids may not be made
sufficiently rapidly and would need to be sup-
• transamination of amino acids, conserving plied in the diet. The exact requirements of
their nitrogen as the a-amino group of glu- non-ruminant animals for dietary amino acids
tamate, and degrading the carbon skeletons; are rather complex. They depend on the
• transferring the amino group of glutamate species, its physiological state and other com-
to a new range of a-ketoacids to form a new ponents of the diet. For example, the need for
spectrum of amino acids. methionine can be reduced by supplying cys-
teine, and that for tyrosine by supplying
This process takes place in all organisms. It phenylalanine as, in each case, the former can
permits them to make best use of the amino be made from the latter.
acids available in the diet, and it allows them The synthesis of proteins requires a supply
to reprocess amino acids formed by protein of all their constituent amino acids in the cor-
turnover to match the changing demands of rect proportions. Rates of protein synthesis will
protein synthesis. In non-ruminant animals, therefore be determined by availability of the
however, the amino acids synthesized as a limiting amino acid, i.e. the one present in
result of these interconversions are limited to smallest quantity in relation to the demands of
the non-essential ones. the cell. In general the balance of amino acids
Although bacteria are capable of making all present in animal-derived foods is closer to the
the amino acids, they often do not synthesize dietary requirements of animals than the bal-
a particular amino acid if it is available in the ance in most plant-derived foods. Cereal-based
environment. Normally the presence of the diets tend to be deficient in lysine, methionine,
amino acid prevents the synthesis of all the threonine and tryptophan, so some or all of
enzymes required for its biosynthesis, by these need to be obtained from other compo-
switching off their genes. This is an example of nents of the diet. In non-ruminant diets, lysine
the process known as enzyme repression, is usually the first limiting amino acid.
which prevents wasteful synthesis of materials
which are not required. This process is
20.7 HERBICIDES AND AMINO ACID
described in more detail in Chapter 21.
BIOSYNTHESIS
Non-ruminant animals must have an ade-
quate supply of all the essential amino acids in An interesting feature of the pathways of
the diet. Ruminants, however, contain in their amino acid biosynthesis in plants is that they
are the sites of action for a number of herbi- • glyphosate, which inhibits conversion of
cides. A number of very potent herbicides shikimic acid into chorismic acid catalysed
inhibit specific steps in the synthesis of amino by the enzyme 5-enolpyruvylshikimic acid-
acids in plants. As the metabolic pathways 3-phosphate (EPSP) synthase, and therefore
affected do not take place in animals, these prevents synthesis of the aromatic amino
herbicides should be of intrinsically low ani- acids (Figure 20.5).
mal toxicity. Herbicides which act in this way
These herbicides are non-selective and kill
include:
crop plants as well as weeds. Genetic engi-
• the sulphonylureas (e.g. chlorsulphuron) neers are interested in inserting genes into
which inhibit acetohydroxy acid synthase, crops to make them resistant to these herbi-
the enzyme which catalyses the initial step cides, as this would provide a very effective
in the synthesis of aliphatic amino acids means of controlling the growth of all weeds
(Figure 20.7); in a crop.
THE SYNTHESIS OF NUCLEIC ACIDS AND 21
PROTEINS

21.2 Synthesis of purine and pyrimidine 292
nucleotides
21.3 Replication of DNA 292
21.3.1 DNA synthesis in viruses 294
21.3.2 Accuracy of DNA synthesis 296
21.4 Synthesis of RNA 296
21.5 The genetic code 297
21.6 Protein synthesis 297
21.6.1 Amino acid activation 297
21.6.2 Initiation 299
21.6.3 Elongation 300
21.6.4 Termination 300
21.6.5 Post-translational modification of 300
proteins
21.6.6 Location of protein synthesis 301
21.7 Regulation of protein synthesis 303
21.7.1 Regulation in prokaryotes 303
21.7.2 Regulation in eukaryotes 305
21.8 Protein synthesis in chloroplasts and 306
mitochondria
21.9 Genetic engineering 308
21.9.1 Enzymes used in DNA manipulation 308
21.9.2 Isolation and synthesis of DNA 308
21.9.3 Gene cloning 309
21.9.4 Screening techniques 310
21.9.5 Restriction fragment length 310
polymorphism (RFLP)
21.9.6 Antisense RNA 310
21.9.7 Vectors and methods for insertion of 310
DNA into cells
21.9.8 Site-specific mutagenesis 311
(oligonucleotide-directed mutagenesis)
21.1 INTRODUCTION part of the protein synthesizing machinery of

the cell. The synthesis of either type of nucle-
DNA is synthesized as the chromosomes ic acid requires a supply of the purine and
replicate prior to cell division. RNA is made pyrimidine nucleotides of which they are
continuously, although at varying rates as composed.
292 The synthesis of nucleic acids and proteins
21.2 SYNTHESIS OF PURINE AND 21.3 REPLICATION OF DNA
PYRIMIDINE NUCLEOTIDES
The replication of double-stranded DNA takes
The structures of the purines and pyrimidines place during the S-phase of the cell cycle by a
commonly found in nucleic acids are described semi-conservative mechanism. In this, the two
in Chapter 7. The purines are synthesized as strands separate and copies of each are made
shown in Figure 21.1. Synthesis starts from in such a way that each daughter cell inherits
ribose-5-phosphate which is converted to one of the original strands and a copy of the
phosphoribosyl pyrophosphate (PRPP) by other DNA strand (Figure 21.3).
attachment of a pyrophosphate group derived The first stage in DNA synthesis is separa-
from ATP. The purine ring structure is then tion of the DNA strands. Because of the com-
gradually built up by reaction with glutamine, plex manner in which they are twisted
glycine, 10-formyl tetrahydrofolate, further around one another, only a part of the chains
glutamine, ATP, carbon dioxide and more separates at anyone time and forms a repli-
formyl tetrahydrofolate. The product of the cation fork or replicon. This is aided by the
pathway is inosine-5' -monophosphate (IMP) presence of unwinding enzymes called heli-
in which the purine ring is already attached to cases.
the ribose sugar. Guanosine-5' -monophos- DNA is synthesized by the enzyme DNA
phate (GMP) is formed from IMP by oxidation polymerase. There are several forms of this
and reaction with glutamine, whilst AMP is enzyme in both prokaryotes and eukaryotes.
produced by reaction with aspartate. The Some polymerases appear to be involved in
purine nucleoside monophosphates are con- DNA replication and others in DNA repair.
verted to triphosphates by transfer of phos- DNA polymerase can only make DNA from
phates from ATP. the 5' to the 3' end. Because DNA is double-
Pyrimidines are synthesized in an entirely stranded and the chains run in opposite direc-
different way, the ring being assembled tions, it is not possible for both strands to be
before being linked to the sugar. Synthesis replicated simultaneously in the same direc-
starts with formation of carbamoyl phosphate tion. The new strand which needs to be made
from ATP, carbon dioxide (as bicarbonate) from the 5' to the 3' end (the leading strand)
and glutamine. Reaction with aspartate gives can be made continuously and in one piece.
carbamoyl aspartate which cyclizes to form However the other chain (the lagging strand)
dihydroorotate and then orotate. This reacts must be replicated in short pieces, each of
with PRPP to form orotidine-5' -phosphate which is made in the 5' to 3' direction, and
(OMP), which is decarboxylated to UMP which are later joined together (Figure 21.4).
(Figure 21.2). Reaction of UMP with ATP These pieces are called Okazaki fragments
forms UTP, from which cytidine 5' -triphos- after their discoverer. Okazaki fragments in
phate (CTP) is synthesized by reaction with bacteria may be up to about 2000 bases long,
ATP and glutamine. but those found in eukaryotes are usually only
Deoxyribonucletoides required for DNA about 150 bases long.
synthesis are made, in most organisms, by DNA polymerase requires a short piece of
reduction of the corresponding ribonucleotide 'primer' RNA, about 10 nucleotides long,
diphosphates using thioredoxin (a small, sul- made by an enzyme called primase on which
phur-containing protein) as the reducing is assembled the DNA. The RNA is later
agent. Formation of thymidine is brought removed and replaced by DNA by another
about by methylation of dUMP, using 5,10- form of DNA polymerase.
methylenetetrahydrofolate as the source of Both chains of the DNA molecule are repli-
the methyl group. cated at the same time. In prokaryotes this is
Replication of DNA 293
aspartate
• IIT'".......... 10-formyl-
tetrahydrofolate
10-formyl-
tetrahydrofolate
QcAV®
®OCH 2 0
ATP ADP
OH O"'(9lutamine
Ribose-5-phosphate PRPP
glutamate
o
glycine
10-f0rmyl @)oCH2 0 NH2
tetrahydrofolate
ATP
OH OH
c~ 5-ph0sph0ribosylamine
10-formyl
tetrahydrofolate
AMP
GMP
Figure 21.1 Pathway for the biosynthesis of purines. The origin of the atoms in the purine ring is indicat-
ed in the upper part of the figure with an outline of the pathway below.
done by a single DNA polymerase molecule which is being replicated, it actually runs
with two active sites, but in eukaryotes a sepa- through the enzyme in the same direction as
rate DNA polymerase catalyses formation of the leading strand.
each chain. In prokaryotes the lagging strand Once the sections of DNA at each replicon
may coil around so that, over the section are complete they are joined together through
-.~-
carbamoyl
phosphate
NHz aspartate Pi NH2
U
Glutamine 6=0 6=0
+
2ATP 6 ~H
+
HC03- -oJ..--o .J:-COOH
6- 6H2
~/
600H
phosphate
N-carbamoyl
aspartate
Orotate Orotidine-5- UMP

phosphate
Figure 21.2 Pathway for the biosynthesis of pyrimidines. The atoms of the pyrimidine ring all originate
from aspartate and carbamoyl phosphate.
the action of DNA ligase to form continuous dNTP + (dNMP)n -+ (dNMP)n+l + PPj
DNA molecules.
where dNTP and dNMP represent any nucleo-
In prokaryotes such as Escherichia coli, repli-
side tri- or monophosphate, respectively.
cation of DNA starts at one location and pro-
Mter synthesis, some modification of the
gresses in both directions (hi-directional). The
DNA may take place, e.g. some of the cytosine
origin, where replication begins, may be rich
bases may be methylated, and this may playa
in AT base pairs which are relatively easily
role in regulation of the expression of some
separated due to weak hydrogen bonding
genes.
(Chapter 7)- In contrast, in eukaryotes there
appear to be multiple origins at which DNA
synthesis may begin. There are therefore
21.3.1 DNA SYNTHESIS IN VIRUSES
many replication forks.
DNA polymerase catalyses the following The genetic material in viruses can be single-
reaction: or double-stranded DNA or RNA. Some virus-
Replication of DNA 295
Figure 21.3 Replication of double-stranded DNA. Each daughter ceIl receives one of the original strands
and a newly made strand which is complementary to this.
3'
leading strand
5'
3'
IHelicases
lagging strand
5'
Figure 21.4 Replication of double-stranded DNA catalysed by DNA polymerase. The leading strand is
synthesized in a continuous piece, but the lagging strand is made in short pieces which are subsequently
joined together.
es carry the genetic information to code for the arranged in such a way that three bases in
enzymes needed for replication of their DNA, DNA or mRNA determine the nature of one
but others rely on the host's enzymes for this amino acid. This is therefore called a triplet
purpose. Some viruses contain RNA as their code.
genetic material, e.g. tobacco mosaic virus, The flow of information is thus:
influenza virus, polio virus. In certain types of
DNA ---> RNA ---> Protein
RNA virus, e.g. some cancer-causing retro-
viruses and the AIDS virus, an enzyme called
transcription translation
reverse transcriptase synthesizes DNA using
the RNA as a template. The DNA produced is The transfer of information from DNA to
then incorporated into the host DNA, leading RNA is known as transcription, and that from
to synthesis of new virus particles. Another RNA to proteins as translation. As the message
virus, hepatitis B, contains DNA as genetic in DNA and in RNA exists in the same triplet
material but uses RNA as an intermediate in its form, it simply has to be 'transcribed' from one
replication. to the other. However, during protein synthe-
sis the triplet sequence of bases has to be trans-
21.3.2 ACCURACY OF DNA SYNTHESIS lated into the sequence of amino acids.
RNA is synthesized by RNA polymerases
Several mechanisms ensure that the sequence
which catalyse the reaction:
of bases in DNA is replicated extremely faith-
fully. Some types of DNA polymerase have NTP + (NMP)n ---> (NMP)n+! + PP j
the capacity not only to add bases to the 3' RNA polymerase can make RNA only by
end of the growing chain (polymerase activi- starting at the 5' end and moving towards the
ty) but also to remove them (depolymerase 3' end. It does this by sequentially inserting
activity). If an incorrect base is inserted it is bases which are complementary to those in
unable to form a base pair with the template, one chain of the DNA. All types of RNA are
and the depolymerase activity of the enzyme made by this reaction, although different
is increased so that the incorrect base is forms of RNA polymerase may be responsible
removed before further bases are added. This for the formation of mRNA, tRNA and rRNA.
process is referred to as 'editing' or 'proof- The RNA which is made by RNA poly-
reading'. merase is called the primary transcript and
In both prokaryotes and eukaryotes, the may undergo further processing before it
main function of some DNA polymerases reaches its final, functioning state. In eukary-
seems to be to repair DNA. In E. coli, Pol III otes this may involve removal of introns,
appears to be the main enzyme catalysing syn- addition or removal of other groups, or mod-
thesis of DNA, whilst Pol I seems to be con- ification of some of the bases (see section
cerned mainly with repair processes. There are 21.7.2). In bacteria, mRNA does not undergo
also mechanisms for repair of damaged DNA further processing but precursors of tRNA
resulting from exposure to UV light,.ionizing and rRNA do.
radiation or chemical mutagens.
Processing of mRNA
21.4 SYNTHESIS OF RNA
The product of RNA polymerase II is called
The sequence of amino acids in proteins is heterogeneous nuclear RNA (hnRNA)
determined by the sequence of bases in because it contains molecules of a variety of
mRNA, which in turn is determined by the sizes. This must be processed to yield mRNA
sequence of bases in DNA. The information is proper.
Protein synthesis 297
Attachment of 3' and 5' caps eukaryotes; only about 5% of the total RNA
transcribed leaves the nucleus .
• 5' -end: the first-made 5' -end is capped' by
I
attaching to it a special group. Capping

occurs before transcription is complete. The 21.5 THE GENETIC CODE
cap is 7-methylguanosine triphosphate and
Information required for protein synthesis is
the next residue also has a 2' methyl group
carried to the ribosomes, where protein syn-
attached to the 2' OH of its ribose group
thesis takes place, by mRNA molecules. A
(Figure 21.5).
group of three adjacent bases in the mRNA
• 3' -end: after transcription is complete the
determines the nature of each amino acid in
enzyme polyA-polymerase adds 100-200
the protein. Given that there are four bases -
residues of adenylic acid (polyA) to the 3'
adenine (A), cytosine (C), guanine (G) and
end of mRNA molecules. The polyA tail
uracil (V) - there are 64 possible different com-
appears to mediate subsequent RNA pro-
binations of three of these, each of which
cessing and transport from the nucleus to
could code for one amino acid. For example
the cytoplasm. No polyA sequence is added
the sequence AVG in mRNA will result in
to the mRNA in prokaryotes.
incorporation of a molecule of methionine into
Transcripts produced by RNA polymerase a protein. Not all triplets code for an amino
II are generally unstable and short-lived. The acid, and the code is degenerate as more than
stability of mRNA may be related to the length one triplet may code for some amino acids.
of the polyA tail as older mRNA molecules Where this is the case, these degenerate
have shorter tails. codons normally differ only in the last base
(see Chapter 31).
The genetic code shown in Figure 21.6 is
Removal of introns (RNA splicing)
almost universal, although minor variations
lntrons are regions in mRNA which do not are found in some very simple organisms and
code for amino acids and which are removed in mitochondria.
or excized from the RNA before it is used in
protein synthesis. Introns vary in size between
21.6 PROTEIN SYNTHESIS
about 100 and 10 000 nucleotides or more.
There can be very wide differences in the Protein synthesis is the assembly of amino
sequence of bases present in different introns, acids into a linear sequence using mRNA as a
but the few nucleotides at each end are nearly template. The process can be divided into four
the same in all of them. The sequence has GV parts: amino acid activation, initiation, elonga-
at the 5' end and AG at the 3' end. This region tion and termination.
seems to be complementary to a sequence in
the RNA contained in a particle called a small
21.6.1 AMINO ACID ACTIVATION
nuclear ribonuclear particle VI (snRNP).
These take part in RNA splicing by forming an Before amino acids can be assembled into pro-
RNA-RNA helix with the ends of the introns, teins, each must be attached to the correspond-
so that they are brought together. They can ing tRNA molecule to form an aminoacyl
then be cleaved and resealed (Figure 21.5). tRNA. This takes place as shown in Figure 21.7.
Intron removal is not very species-specific. These reactions are catalysed by enzymes
Thus the five introns in bean phaseolin RNA called amino acyl tRNA synthetases (or amino
are correctly removed in tobacco. However, acid-activating enzymes). Activation takes
although yeast will carry out some intron exci- place in two stages: first the amino acid reacts
sion this is often not the same as in higher with ATP to form an aminoacyl adenylate,
(a)
5' 3'
T
5'cap -
7-methy/guanosine
Introns
T3'cap -
polyA
triphosphate
(b)
Small rtbonuclea,
particle (U1)
Proleln --j'-1----l~_
Figure 21.5 (a) The location of caps and introns in eukaryotic hnRNA. (b) Mechanism for removal of
introns by snRNP.
which then reacts with the tRNA molecule to acid. Each amino acid becomes attached only
form the aminoacyl tRNA. Both reactions are to its own tRNA molecule (e.g. alanine to
catalysed by the same enzyme. There is at least tRNAa1a ). This tRNA molecule has an anticodon
one tRNA and one enzyme for each amino complementary to the triplet of bases in mRNA
1st position 2nd position 3rd position
U C A G
Phe Ser Tyr Cys U

U Phe Ser Tyr Cys C
Leu Ser STOP STOP A
Leu Ser STOP Trp G
Leu Pro His Arg U

C Leu Pro His Arg C
Leu Pro Gin Arg A
Leu Pro Gin Arg G
lie Thr Asn Ser U

A lie Thr Asn Ser C
lie Thr Lys Arg A
Met Thr Lys Arg G
Val Ala Asp Gly U

G Val Ala Asp Gly C
Val Ala Glu Gly A
Val Ala Glu Gly G
Figure 21.6 The genetic code. This code is almost universal in all organisms.
which codes for alanine. It is essential that protein synthesis begins at an AUG sequence
attachment of the amino acids to the tRNA is which is preceded by a purine-rich region 10
extremely accurate as any errors here will lead nucleotides away (the Shine-Dalgarno
to the insertion of an incorrect amino acid into sequence) which binds to the small subunit of
the protein, with potentially disastrous conse- the ribosome (Figure 21.8).
quences. Protein synthesis takes place on the ribo-
somes, which are small subcellular compo-
nents present either free in the cytoplasm or
21.6.2 INITIATION
attached to the membranes of the endoplas-
Proteins are synthesized starting from the N- mic reticulum. In either case they consist of a
(amino) terminal end (Chapter 5) with a deriv- small and a large subunit (see Chapter 7).
ative of methionine as the first amino acid in Between the two subunits there is a groove
their sequence. In prokaryotes this is N-formyl into which a mRNA molecule fits. There are
methionine (f-MET) but in eukaryotes it is also two sites at which aminoacyl-tRNA mole-
methionine itself (MET). In both cases the cules can be attached to the ribosomes. One of
codon in the mRNA which codes for these these is called the A site and the other the P
amino acids is AUG. In eukaryotic cells, protein site (Figure 21.9).
synthesis usually begins at the AUG nearest In the initial step in protein synthesis, a
the 5' end of the mRNA. In prokaryotic cells, complex is formed between the mRNA, Met-
base-pairs to the second codon in the mRNA.
The enzyme peptidyl transferase (part of a
ATP large ribosomal subunit) now catalyses the for-
mation of a peptide bond between the two
amino acids which remain attached to the
tRNA at the A site. The ribosome moves one
triplet along the mRNA so that the growing
peptide and its tRNA are at the P site and the
A site is empty. GTP is needed for this process,
H 0 0 PPi which is called translocation (Figure 21.9).
I i I
NHr C- C- 0- P- O-ribose-adenine
I I 21.6.4 TERMINATION
R H
Elongation continues until one of the 'stop'
Arninoacyladenylate codons is reached on the mRNA. These
co dons are UAA, UAG or UGA. When the ribo-
some reaches one of these, the bond holding
the growing polypeptide to the tRNA is bro-
ken and the polypeptide is released. The tRNA
and ribosomal subunits dissociate and are
ready to start synthesizing another protein.
H 0
Because mRNA molecules are quite long,
I I
NHr C- C- 0- tRNA several ribosomes can synthesize proteins on
I each mRNA at one time. Under the electron
R microscope, complexes of this type have the
Arninoacyl tRNA appearance of beads on a string and are called
polysomes. There can be one ribosome
Figure 21.7 Formation of aminoacyl tRNAs. The
two-step reaction takes place on the aminoacyl approximately every 80 bases along the
tRNA synthase enzymes and produces an amino- mRNA, so about five ribosomes can operate at
acyladenylate as an intermediate. once on the mRNA for a protein such as
haemoglobin (500 nucleotides). This speeds up
protein synthesis considerably.
tRNAmet (or f-Met-tRNA f_met), several protein The equivalent of at least four ATP mole-
initiation factors, and the small subunit of the cules are used in the synthesis of each peptide
ribosome. The large subunit also attaches itself bond. In many cells, more energy is used in
to complete the complex. The anticodon protein synthesis than in any other process.
region of the tRNA molecule forms a base pair
with the AUG codon of the mRNA and is held
21.6.5 POST-TRANSLATIONAL MODIFICATION
at the P site on the ribosome. Formation of this
OF PROTEINS
complex requires GTP.
After release from the ribosome, protein mole-
cules may be modified before they reach their
21.6.3 ELONGATION
final, 'mature' form. In many proteins, partic-
A second tRNA carrying the amino acid corre- ularly in eukaryotes, some of the N-terminal
sponding to the second codon on the mRNA residues are removed by hydrolysis after syn-
attaches itself to the A site on the ribosome, thesis is complete. Thus, although all proteins
and GTP is hydrolysed. This tRNA molecule are synthesized with Met or f-Met as the N-
(a)
AUG
(b)
-10 +1
i
AUG
Shine-Dalgamo
sequence
Figure 21.8 mRNA sequences which are involved in initiation of protein synthesis. (a) In eukaryotic cells
the AUG nearest the 5' end of mRNA serves as the start codon for synthesis of the protein. (b) In
prokaryotic cells start AUG codons are preceded by the Shine-Dalgamo sequence, which is rich in
purines and is complementary to part of the RNA in the ribosomes.
terminal amino acid, many mature proteins do teine residues and act as solid 'anchor' points
not have these residues at their N-terminus. in the structure of proteins, stabilizing their 3-
Sugar or phosphate residues may be added to D shape. Inappropriate folding of proteins dur-
proteins after they have been synthesized. ing synthesis may be prevented by formation
Addition of sugar residues to form glycopro- of complexes with chaperone proteins.
teins is particularly common in the case of pro-
teins which will be secreted from the cell or
21.6.6 LOCATION OF PROTEIN SYNTHESIS
incorporated into membranes. In addition,
some amino acid side chains may be modified, The sequence of events described above leads
e.g. proline and lysine may be hydroxylated to to the release of proteins into the cytoplasm.
form hydroxyproline and hydroxylysine, Enzymes involved in glycolysis, gluconeogen-
respectively, as in collagen (see Chapter 30). esis, the pentose phosphate pathway, etc. are
Proteins appear to be folded into their made in this way. In some cases, however,
native conformation by a sequential mecha- mechanisms are required to ensure that pro-
nism which begins during their synthesis. teins are targeted to other parts of the cell, or
Regularly repeating structures such as pleated even outside the cell.
sheet or a-helix are formed first, followed by Proteins which are to be secreted from the
organization of domains covering larger areas, cell or packaged into protein bodies (see
and then finally combination of these domains Chapter 23) are synthesized with short N-ter-
to yield the native conformation. As the pep- minal pre-sequences or signal peptides of
tide chain folds, disulphide (-5-5-) bridges are between 13 and 36 amino acids. The signal
formed between the -SH groups of nearby cys- peptide is hydrophobic and usually has a basic
GOP
Pi
Peptidy/
transferase
GOP GTP
Pi
Translocation
Figure 21.9 The steps concerned in protein synthesis in prokaryotes. In prokaryotes AUG signals the N-
terminal amino acid which is f-Met. In eukaryotes, Met is inserted in the N-terminal position.
region near to its N-terminal end. As soon as lumen side of the membrane. Proteins made in
the signal peptide has been synthesized it this way are thus released into the lumen of
forms a complex with a signal recognition par- the ER. They are then packaged by the Golgi
ticle (SRP) which contains RNA and protein, bodies into vesicles which fuse with the plas-
and the complex binds to the membranes of ma membrane, releasing their contents to the
the endoplasmic reticulum (ER). As the pep- outside of the cell. Many proteins are made
tide grows it passes through the membranes and secreted in this way, including those
into the lumen or sac of the reticulum, where found in blood serum, milk, cell-wall proteins,
it forms a complex with chaperone proteins glycoproteins on the cell surface, digestive
which prevent it from folding. During trans- enzymes and protein hormones. These are
port through the ER membranes, the signal often glycoproteins, i.e. they have sugar
peptide is cleaved by signal peptidases on the residues attached. A further function of the ER
Regulation of protein synthesis 303
membrane is to attach these carbohydrates, level. This, in combination with the much larg-
most commonly to asparagine residues in the er genome and the existence of differentiated
protein. cells, results in patterns of regulation which
Some proteins pass, after synthesis, to the are extremely complex.
mitochondria or chloroplasts. Such proteins
are synthesized with pre-sequences called
21.7.1 REGULATION IN PROKARYOTES
transit pep tides at their N-terminal ends, but
in this case they are synthesized by ribosomes In E. coli and other bacteria, a single type of
which are free in the cytoplasm rather than RNA polymerase is responsible for the synthe-
attached to the ER. The transit pep tides typi- sis of mRNA, tRNA and rRNA. Although made
cally consist of 15-35 residues and are rich in by the same enzyme they are synthesized at
basic and hydroxylated amino acids such as different rates, so that they are produced in
serine and threonine. the correct quantities according to the
The information in these transit peptides is demands of the cell.
sufficient to target them accurately not just to RNA polymerase begins to synthesize RNA
the correct organelle, but to individual com- at specific locations on the DNA molecules.
partments within it, such as the mitochondrial These are recognized because of the existence
matrix or the space between the chloroplast of 'promoter sites' which lie upstream of the
membranes. start of the coding sequence in the DNA. Two
Folded proteins are not easily transported sites within this promoter region are impor-
through membranes, and folding is prevented tant in controlling attachment of the RNA
by binding of the peptide to chaperone pro- polymerase and initiation of RNA synthesis.
teins both before and after transport across the These are centred 35 and 10 base pairs before
membrane. This ensures that folding takes the start of the coding sequence (positions -35
place only when the entire peptide has and -10, respectively). They determine where,
crossed the membrane. how strongly, and how frequently RNA poly-
All of the information needed to determine merase initiates transcription of the structural
the destination of proteins in the cell is con- genes which they control.
tained within the N-terminal peptides. It is The features of promoter sites which are
thus possible to direct proteins to specific loca- common to different bacteria are summarized
tions simply by adding suitable pre-sequences in Figure 21.10. At the -35 position the com-
to their genes. monest sequence is TTGACA, and at -10 it is
TATAAT. The latter is known as the Pribnow
or TATA Box. Many promoters differ in one or
21.7 REGULATION OF PROTEIN SYNTHESIS
more positions from these 'consensus'
In prokaryotic organisms, protein synthesis is sequences, and generally the better the match
regulated mainly at the level of transcription, the more efficient the promoter.
i.e. mRNA synthesis is controlled. Because RNA polymerase consists of 'core' enzyme
mRNA synthesis and protein synthesis take (itself composed of several subunits) and a
place in the same cell compartment, the protein subunit called (T70 which recognises
processes occur in rapid succession. Indeed, promoter sequences. Variants of (T70 may
synthesis of proteins may even begin using replace it under certain circumstances, and
incomplete mRNA molecules. There is there- each of these may recognise different promot-
fore no time for complex modification of er sequences, allowing transcription of differ-
mRNA. In eukaryotes, on the other hand, in ent sets of genes. Sudden changes in tempera-
addition to transcriptional control, there is the ture cause the production of different (T sub-
opportunity for control at post-transcriptional units which change the specificity of RNA
Pribnow or TATA Structural genes
box
Pos~ion -35 -10 +1
I I ITITIGWCIAI I I I 1::::::::::::1 11*1**1 I III·.·.··.·.·.·.·.··.·.·, I II I II I I I II [ ................... J I II 1111 II II I DNA

STOP
IA!u!31 II II mRNA
Figure 21.10 RNA polymerase binding sites to prokaryotic DNA. The structural genes beginning at posi-
tion + 1 are preceded by promoter regions around 10 and 35 base pairs upstream.
polymerase, resulting in the production of tosidase must be made. The amount of the
heat-shock proteins. Changes in a subunits enzyme increases very rapidly so that within
also occur in response to variation in nitrogen 1-2 minutes there may be 5000 molecules per
sources, activating genes required for nitrogen cell. The activity of two other enzymes ([3-
assimilation. galactoside permease and [3-thiogalactoside
The rate at which the basic transcription transacetylase) also rise at the same time. The
process occurs can be modified in a great num- manner in which this operon functions is
ber of ways. Thus there may be attenuators', I shown in Figure 21.1l.
preceding structural genes which cause the The three enzymes are coded by three
synthesis of mRNA to be abandoned after only structural genes which are adjacent to one
a short leader piece has been synthesized. another. All are transcribed to form a single
These are common, for example, in the genes piece of mRNA. An operator, which is located
coding for enzymes involved in bacterial syn- next to the structural genes, is able to bind
thesis of amino acids. tightly to a protein called the repressor pro- I
tein'. When it does so, it inhibits binding of

RNA polymerase and prevents transcription
Operons
of the three structural genes.
In bacteria, the genes required for a metabolic When an inducer (in this case allolactose, a
pathway often occur in the form of an operon. product of lactose metabolism) is present, it
This consists of the genes which code for the binds to the repressor protein and causes a
enzymes catalysing the process (structural change in conformation. This prevents it from
genes) together with genes coding for products binding to the operator gene, so that tran-
which regulate the expression of the structural scription of the structural genes can take place.
genes. The lac operon of E. coli has been inten- One operator controls the activity of sever-
sively studied and has provided insights into al structural genes which are adjacent to it.
how many processes are regulated in bacteria. When the operator is unblocked, the single
The enzyme [3-galactosidase catalyses the piece of mRNA is produced and all three
hydrolysis of sugars such as lactose, which enzymes are synthesized. Enzymes which are
contain [3-galactoside bonds. It converts lac- synthesized only when needed, like those
tose into glucose and galactose as an essential described here, are called inducible enzymes.
first step in lactose metabolism. When E. coli is Other systems which are regulated in a sim-
grown in the presence of glucose as a carbon ilar manner also occur in bacteria. If E. coli is
source, the levels of this enzyme are very low grown in a medium containing NH4 + and a
(about five enzyme molecules per cell) as it is carbon source it normally synthesizes the
not needed. If the glucose is withdrawn and enzymes needed for synthesis of each of the 20
lactose is provided as a carbon source, [3-galac- amino acids. However when one of the amino
Regulation of protein synthesis 305
Structural Structural Structural

Regulator Operator gene 1 gene 2 gene 3
DNA
polycistronic
mRNA
1 1 !
Proteins
RepreuOT protein
normally binds to
•
and blocks operator
gene.
Binding of inducer to
repressor protein
prevents it blocking
operator gene
Figure 21.11 The lac operon in Escherichia coli. Transcription of the structural genes is blocked by binding
of a repressor protein to the operator gene. Binding of an inducer to the repressor protein prevents its
attachment to the operator, allowing the structural genes to be transcribed.
acids (e.g. histidine) is provided in the nutrient proteins with related functions (e.g. the
solution, all the enzymes needed for its enzymes catalysing steps in a metabolic path-
biosynthesis disappear. way) tend to be closely clustered together on
The regulation of this system is very similar the DNA of prokaryotes, those in eukaryotes
to that of the lac operon, but in this case the are often distributed widely amongst the chro-
repressor protein is only able to combine with mosomes. Within chromosomes the DNA is
the operator gene after binding to a co-repres- tightly complexed with proteins which modify
sor (histidine or a derivative). When histidine expression of the genes.
is present it switches off synthesis of all the The specialized cell types in eukaryotes, e.g.
structural genes in the operon. This is called liver, mammary and brain cells, differ from
'enzyme repression'. one another because they contain different
Enzyme induction and enzyme repression proteins. Although all cells of an organism
are very common in bacteria. These organisms contain identical DNA, only a small part of the
encounter frequent changes in nutrient sup- total DNA is expressed in anyone cell. In dif-
ply, and mechanisms such as these enable ferent cell types, different parts of the DNA are
them to make efficient use of resources. active. Switching on or off individual genes
forms the basis of the process of differentiation
or specialization of cells in multicellular organ-
21.7.2 REGULATION IN EUKARYOTES
isms.
In eukaryotes, the genes are arranged on a This complexity means that the manner in
series of chromosomes. Whereas genes for which the activity of the eukaryotic genome is
regulated is not nearly as well understood as 21.8 PROTEIN SYNTHESIS IN
in prokaryotes. However, the following are CHLOROPLASTS AND MITOCHONDRIA
thought to be important in activating and
Mitochondria and chloroplasts contain DNA of
inactivating genes:
their own and can carry out protein synthesis.
• changes in the distribution and packing of The DNA in both chloroplasts and mitochon-
nucleosomes; dria is roughly circular and double-stranded,
• methylation of DNA bases; and there are multiple copies of the DNA mole-
• chemical modification (acetylation, methy- cules. No histones are associated with the DNA
lation and phosphorylation) of his tones in these organelles. In most animals, organelle
associated with the DNA; DNA (mitochondrial) makes up less than 1% of
• changes in patterns of non-histone pro- the total DNA of the cell, but in green plant tis-
teins. sues organelle DNA (mitochondrial and chloro-
plast) can be up to 15% of the total DNA.
In eukaryotes, RNA synthesis is catalysed
Although these organelles are able to syn-
by three different types of RNA polymerase.
thesize their own proteins, only a few arise in
RNA polymerase I forms rRNA; RNA poly-
this way. Most organelle proteins are encoded
merase II forms large molecules of RNA
which give rise to mRNA, and RNA poly- in the nuclear genome, made in the cytoplasm
and then transported to the organelle. Details
merase III forms small RNA molecules such
as tRNA and the small components of the of the processes for which the organelle
ribosomes. genomes are responsible are given in Table
The genes in eukaryotes do not occur as 21.1.
operons. Each coding region has its own regu- The genome of mitochondria codes for
latory sequences. In addition, as discussed tRNAs, rRNAs and about 20 proteins, includ-
below, the coding sequences are often inter- ing some of the inner mitochondrial mem-
rupted by non-coding sections which are brane (Table 21.1). Although very variable
removed during RNA processing. between species, there is much non-coding
There are specific promoter sequences DNA. Mitochondrial mRNA has no 5' -caps and
upstream of the start of the coding region of no extensive 3' -polyA tails, but it may contain
eukaryotic genes. The best characterized of introns. Protein synthesis in mitochondria
these is the TATA or Hogness box which is resembles that in bacteria. Synthesis of proteins
about 25 bases upstream of the transcriptional starts with f-Met, takes place on 70S ribosomes,
start. The consensus sequence of this is and shows similar sensitivity to antibiotics.
TATAAA. This sequence appears to direct The chloroplast genome is much more com-
RNA polymerase to the correct AUG codon at plex than that of the mitochondria: a large
which to start transcription (Figure 21.12). number of proteins are made in this organelle.
Between about 40 and 110 bases upstream of The DNA exists as double-stranded, circular
the transcriptional start are found sequences molecules. The synthesis of RNA in chloro-
which regulate the frequency of initiation of plasts is thought to be very like that occurring
transcription of the gene. In animals there are in bacteria: there are no 5' caps or 3' polyA
commonly two of these, called the GC and sequences, and very little processing of RNA
CAAT boxes, but in plants the latter is often takes place. Protein synthesis is initiated by f-
absent or replaced by alternatives (e.g. CATC Met and resembles bacterial protein synthesis
box in cereals). Additional enhancer sequences even more closely than does that taking place
which stimulate the activity of nearby promot- in mitochondria. The ribosomes in chloro-
ers may also be found either up- or down- plasts are of the 70S type and are different
stream of the coding sequences. from cytoplasmic ribosomes.
Protein synthesis in chloroplasts and mitochondria 307
TATAor
GCbox CAATbox Structural gene
Hognessbox
-110 -40 -25 +1
DNA
STOP
II mRNA
\ / T
S' cap In/rons 3' poIyA
sequence
Figure 21.12 Promoter sequences in eukaryotes. Structural genes are preceded by promoter regions 25, 40
and 110 base pairs upstream.
In some cases, some subunits of complex unit is coded for by the nucleus and the large
proteins are coded by nuclear and some by one by the chloroplast, and they are assem-
chloroplast genes. Rubisco is the best known bled together in the chloroplast to produce
of such proteins. In this case the small sub- active protein.
Table 21.1 Protein synthesis in organelles
Functions Proteins synthesized
Mitochondrial functions coded by tRNAs

the mitochondrial genome
rRNAs
about 20-30 proteins: Fll.ATPase (ATP synthesis); subunits of
cytochrome c oxidase; cytochrome b; a ribosomal protein prod-
uct giving rise to cytoplasmic male sterility
Mitochondrial functions coded by TCA cycle enzymes
the nuclear genome
other proteins involved in electron transport and oxidative
phosphorylation
structural proteins
Chloroplast functions coded by chloroplast tRNAs
the chloroplast genome
chloroplast rRNAs
large subunit of Rubisco
cytochromes f. b-559
protein synthesis elongation factors
electron transport coupling factors
some subunits of the proton-translocating ATPase components
of the P7011.chlorophyll-protein complex
Chloroplast functions controlled by chlorophyll synthesis
nuclear genes
chloroplast carotenoids
aminoacyl-tRNA synthetases
70S ribosomal proteins
subunits of the proton translocating ATPase
small subunit of Rubisco
21.9 GENETIC ENGINEERING junction with restriction enzymes, allows
genes to be linked to other DNA to produce
In recent years a wide variety of methods for
the investigation and manipulation of gene recombinant products.
Reverse transcriptase catalyses the forma-
structure have been developed. Collectively,
tion of complementary DNA (cDNA) using
these have allowed a very rapid increase in
RNA as a template. This allows genes to be
understanding of many aspects of gene
obtained from corresponding mRNA mole-
function and have provided the depth of
cules.
knowledge necessary to begin to manipulate
DNA polymerase catalyses polymerization
agricultural systems to increase production
and quality. The success of these methods has of deoxyribonucleoside triphosphates using a
DNA template to produce a complementary
depended on:
copy.
• procedures for identification and sequenc-
ing of genes;
21.9.2 ISOLATION AND SYNTHESIS OF DNA
• methods of synthesizing DNA of speCific
sequences; The structure of genes can be determined by
• methods of producing recombinant DNA direct sequencing of DNA or indirectly from
by cutting and reassembling the molecules; the sequence of bases in mRNA molecules.
• methods of modifying the nucleotide DNA sequencing can be carried out by
sequence of genes; chemically cleaving DNA molecules at specific
• vectors for transferring genes from one bases into fragments of a variety of sizes or,
organism to another, e.g. viruses and plas- more commonly, by interrupting DNA syn-
mids. thesis, by addition of synthetic nucleotide ana-
logues, to produce fragments of different
A number of important concepts are out- lengths. Analysis by gel electrophoresis sepa-
lined below as an introduction to applications rates the fragments on the basis of their size
of genetic engineering in agriculture. and provides a simple visual representation of
the nucleotide sequence in the DNA.
21.9.1 ENZYMES USED IN DNA DNA can be synthesized chemically by
MANIPULATION condensation of individual deoxyribonucleo-
side triphosphates. Appropriate protecting
Restriction enzymes or restriction endonucle- groups and 3' -activating groups are required,
ases are enzymes which cleave both strands of and the growing chain is normally attached
double-stranded DNA at specific sequences of to a solid support to allow easy automation.
nucleotides. They are found in many prokary- In this way, synthesis of oligonucleotides up
otes, where their function is to degrade the 100 units long is readily achieved. Such mole-
DNA of invading cells. The enzymes recognise cules are very useful probes which bind to
symmetrical sequences of four to eight base specific complementary sequences within
pairs in the DNA (Figure 21.13). Restriction DNA. Binding of radioactively labelled
enzymes allow DNA molecules to be cleaved probes is a very sensitive method of identify-
at specific sites to produce fragments of vari- ing genes of known sequence within large
ous sizes. amounts of DNA.
DNA ligases join pieces of DNA together cDNA can be synthesized using mRNA
into longer molecules, especially where the templates. RNA is extracted from a tissue
ends have single-stranded complementary which expresses the gene of interest, and
termini, such as are produced by the action of mRNA is separated from this by chromatog-
restriction enzymes. DNA ligase, used in con- raphy on columns carrying poly(dT) chains
Genetic engineering 309
5' 3' 5' 3'

BamHI Haelll
3' 5' 3' 5'
5' 3' 5' 3'

EcoRI Xholl
3' 5' 3' 5'
Figure 21.13 The specificity of four restriction endonucleases. Both strands of DNA are cleaved at sym-
metrical sites. The points of cleavage are indicated by arrows. Where these are not opposite one another,
products with single-stranded 'sticky ends' are produced.
which pair with the polyA tails on the mRNA. Eukaryotic genes may not be correctly
DNA which is complementary to the RNA is expressed in prokaryotes, which cannot
then synthesized on a poly( dT) primer under remove introns from mRNA. However, eDNA
the action of reverse transcriptase. A comple- reconstructed from mRNA lacks introns and
mentary copy of this DNA chain can be can generally be expressed correctly in
obtained by treatment with DNA polymerase prokaryotic cells.
I from E. coli. These processes yield small Genes can also be cloned by peR. This
quantities of eDNA corresponding to all of method uses oligonucleotide primers which are
the types of mRNA present in the cell, and complementary to the DNA on either side of
the quantities of DNA can be increased by the region to be multiplied (Figure 21.14). New
cloning. DNA is synthesized by extension of these
primers. Double-stranded DNA is denatured at
high temperature to separate the strands. The
oligonucleotides are then allowed to bind to the
21.9.3 GENE CLONING
complementary DNA chains at low tempera-
Gene cloning can be achieved in vectors or by ture, and extended by use of DNA polymerase.
the polymerase chain reaction (peR). In the These steps make up a cycle which can be
former case, the DNA to be copied is inserted repeated to give rise to exponentially increasing
into other, larger pieces of DNA such as those amounts of the required DNA. The process uses
of a virus or plasmid. From there it may be heat-stable DNA polymerase enzyme (Taq)
transferred to, and multiplied in, growing from the thermophilic bacterium Thermus
cells. For example, bacterial cells may be aquaticus, which survives the thermal denatura-
infected by bacteriophages into which eDNA tion of the DNA. Use of peR amplification to
has been inserted. On division this gives rise increase amounts of DNA has allowed analysis
to a cell line or clone containing copies of the and sequencing of very small samples of DNA.
eDNA. A collection of lines representing Thus the root of a single hair may yield, after
eDNA from all the mRNA is called a eDNA amplification by peR, enough DNA for genetic
library. 'fingerprinting' .
single-stranded form. Binding of a radioactive
DNA probe to a copy of the gel, made on a
nitro-cellulose sheet, allows the restriction frag-
ment containing the required sequence to be
identified. Northern blotting is an analogous
region of
interest
technique used for analysis of RNA sequences.
Initial DNA
21.9.5 RESTRICTION FRAGMENT LENGTH
POL YMORPHISM (RFLP)
Denature to separate
chains and anneal to Because restriction enzymes cleave DNA only
primers which Rank the
region of interest at specific sequences of bases, treatment of
DNA with these enzymes results in character-
istic and repeatable fragmentation patterns.
Extend the primers using
DNA polymerase
The fragments are easily separated on the
basis of size by electrophoresis. Mutations
change the fragmentation patterns, which also
vary from one individual of a species to anoth-
Separate chains and
repeat entire process er. Thus, analysis of RFLP patterns may be
used to identify individuals and to locate
genes associated with specific genetic disor-
ders, such as cystic fibrosis.
21.9.6 ANTISENSE RNA

Figure 21.14 The polymerase chain reaction for This is RNA which is complementary to
cloning of DNA. The initial DNA is denatured to mRNA. It forms a duplex with the mRNA and
separate chains and annealed to primers which prevents its translation to produce protein.
flank the region of interest. Extension of suitable
The use of efficient promoter sequences in
primers using DNA polymerase results in synthe-
sis of copies of the DNA of interest. After separa- combination with antisense RNA genes leads
tion of the chains these may be used as templates to the production of antisense RNA which
for synthesis of further copies leading to an expo- may prevent expression of specific deleterious
nential rise in the yield of DNA. genes. Examples of the use of antisense RNA
methodology to delay fruit ripening are
described in Chapter 27.
21.9.4 SCREENING TECHNIQUES
DNA libraries contain large numbers of DNA
21.9.7 VECTORS AND METHODS FOR
sequences, only a few of which are likely to be
INSERTION OF DNA INTO CELLS
of interest, so some form of screening is often
required. This can be achieved by examining Vectors have genomes which can be used to
binding of radioactive probes which are com- introduce DNA into other organisms.
plementary to the DNA sequences of interest Plasmids, bacteriophages and other viruses
(Southern blotting) or by screening the protein and bacteria may be used as vectors. Plasmids
products of the genes with antibodies and A-phage (a bacteriophage which attacks
(Western blotting). the bacterium Escherichia coli) are most com-
In Southern blotting, DNA is degraded by monly used as vectors for cloning DNA in bac-
treatment with restriction enzymes into a large teria. Plasmids are accessory chromosomes
number of fragments, which are separated into found in bacteria and contain circular double-
bands by electrophoresis and denatured to the stranded DNA.
Genetic engineering 311
The DNA of A-phage can become incorpo- 15 nucleotides long is prepared which is com-
rated into the bacterial genome and be repli- plementary to the region of interest, but in
cated with it without killing the bacteria. Large which G replaces C (Figure 21.15). Under par-
parts of the genome of A are not essential for ticular conditions this hybridizes with the nor-
its action and can be replaced by foreign DNA mal gene, as a sufficiently large number of
which is then carried into the bacteria. bases are able to form complementary base
Specifically modified plasmids and bacterio- pairs. The oligonucleotide is then used as a
phages are available which are easier to primer for synthesis of new DNA using the
manipulate and use. For example, they may original as a template. The copy is comple-
have modified restriction sites to allow easy mentary with the exception of the one replace-
insertion of foreign DNA, or carry antibiotic ment. Replication of both strands yields nor-
resistance markers simplifying the selection of mal and modified genes which can be separat-
recombinant organisms. ed and cloned. Examples of the possible uses
Uptake of calcium phosphate-precipitated of this method are increasing the lysine con-
DNA, micro-injection, or genetically modified tent of cereal proteins (see Chapter 23), and
retroviruses, Baculoviruses or Vaccinia viruses changing the specificity of enzymes such as
may be used to insert recombinant DNA into Rubisco (Chapter 17).
animal cells.
Synthetic oligonucleotide
The soil bacterium Agrobacterium tumefaciens
is very commonly used as a vector for inser-
tion of DNA into plant cells. Normally this bac-
terium causes crown galls in dicotyledonous
plants. These arise because of insertion of part
(T-DNA, transferred DNA) of a plasmid called
the Ti plasmid from A. tumefaciens into the
genome of the plant cell. Recombinant DNA
can be incorporated into the plasmid and thus Original DNA
inserted into the plant genome. Electroporetic
insertion of DNA into pro top lasts or bombard-
ment with DNA-coated particles may also be
used for introducing foreign DNA into plant
cells. These methods are especially useful for
plants such as monocotyledons, which are not
infected by A. tumefaciens.
21.9.8 SITE-SPECIFIC MUTAGENESIS (OLIGO-

NUCLEOTIDE-DIRECTED MUTAGENESIS)
Site-specific mutagenesis is a potentially very

powerful and versatile technique for modify-
ing genes. Using this method a single base can Figure 21.15 Site-specific mutagenesis for modifica-
be replaced by any other, leading to replace- tion of base sequences of specific regions of DNA.
ment of one amino acid by another in the pro- Synthetic oligonucleotides are made, with all bases
tein for which the gene codes. As an example, except one complementary to the region of inter-
est. One base may be varied as this does not pre-
serine is coded for by TCT and cysteine by vent base-pairing to the original. The chain is
TGT, so that replacement of serine by cysteine extended using the original as a template. A com-
requires only replacement of a single C by G in plementary copy of this differs from the original
the DNA. To do this, an oligonucleotide about only in the nature of one base.
COMPARTMENTS, MEMBRANES AND 22
REGULATION
22.1 Cell compartments 313

22.2 Lipids and membranes 313
22.3 Transport across membranes 315
22.3.1 Membrane transport mechanisms 316
22.3.2 Other functions of membranes 321
22.4 Principles of metabolic regulation 322
22.4.1 Allosteric regulation 323
22.4.2 Covalent modification 323
22.4.3 Changes in the amount of enzyme 324
22.4.4 Coordinated regulation of pathways 325
22.4.5 Coordination of metabolism in 328
different tissues
22.1 CELL COMPARTMENTS division into numerous compartments within

The major structural features of cells are which specific biochemical processes take
place, in isolation from (but not totally inde-
~escribed in Chapter 1. It is clear that as organ-
Isms have evolved, their architecture has pendent of) processes occurring in other sub-
become more complex, as reflected in both the cellular compartments. Coordination and reg-
ulation of biochemical processes in different
overall shape and size of cells and the diversity
of intracellular structures. Prokaryotes have no subcellular compartments requires communi-
discernible intracellular membranes and can cation across membranes. This may involve
thus be considered as having a single compart- the movement of a substrate or the end prod-
ment. Eukaryotic cells contain many different uct of a pathway from one compartment to
types of membrane-bounded structures. The another, or more complex regulation requiring
the movement of chemical messengers within
plasma membrane surrounds the cell, and acts
as a barrier between the extracellular environ- the cell. Membranes play an important role in
this process, acting as selective permeability
ment which may undergo marked changes in
composition, and the cell contents which have barriers between the different compartments
of the cell, allowing the movement of some
a relatively constant composition. Within cells
molecules but preventing that of others. They
are many subcellular organelles. Many of these
also play an integral part in many intracellular
are surrounded by membranes, e.g. the mito-
chondria, chloroplasts and nucleus, and have signalling pathways and are themselves sites
of a number of metabolic processes.
their own composition which may be markedly
different from the cytosol.
As more has been learned about the bio-
22.2 LIPIDS AND MEMBRANES
chemical processes in complex eukaryotic
cells, it has become apparent that they have The basic structure of all biological membranes
developed metabolic strategies based on their is a lipid bilayer composed mainly of polar
314 Compartments, membranes and regulation
lipids (phospholipids with smaller amounts of teins. Peripheral (or extrinsic) proteins are rela-
glycolipids and sphingolipids). Cholesterol tively loosely attached to the surface of the
and cholesterol esters are also present, but tri- bilayer by polar interactions between amino
acylglycerols are only minor components. The acid side chains and the polar head groups of
physicochemical properties of polar lipids the membrane lipids. They are typical of many
make them particularly well suited as compo- cellular proteins that function in an aqueous
nents of the lipid bilayer. They are amphiphilic environment, in that they are globular proteins
molecules containing both fatty acids which in which the surface amino acids are mainly
are hydrophobic, and polar functional groups polar in nature. Some peripheral proteins are
which are hydrophilic. In the bilayer the fatty covalently linked to the membrane phospho-
acid moieties of these lipids form a central lipid head groups. The second group, integral
hydrophobic core with the polar head groups (or intrinsic) proteins, are embedded in the
on the surface of each face able to interact with lipid layer and may span the entire membrane.
the surrounding aqueous environment. Very few integral proteins have been analysed
Chemical analysis of membranes from vari- for amino acid composition. They are firmly
ous sources has shown that all membranes attached to the membrane and are difficult to
have proteins associated with them. In gener- isolate from other membrane components.
al, the biological activity of a membrane is That part of the protein which protrudes from
reflected in the amount of protein it contains. the membrane surface and is surrounded by
Thus, the myelin sheath surrounding nerve aqueous cell contents has predominantly polar
fibres is composed of approximately 80% lipid amino acids on the surface of the molecule.
and 20% protein. The main function of this However, where the protein passes into the
membrane is to provide a layer of electrical hydrophobic core of the membrane, the amino
insulation around the nerve axon. In contrast, acids are arranged in an a-helical structure
bacterial membranes and the inner mitochon- (Chapter 5) in which the non-polar amino
drial membrane contain about 25% lipid and acids are found on the surface of the protein.
75% protein. These membranes playa role in These amino acids may be attached to mem-
many metabolic processes. The proteins they brane lipids by covalent linkages.
contain have many functions, for example, The most widely accepted model of mem-
some are carriers involved in membrane trans- brane structure is the fluid mosaic model pro-
port, others are structural components of the posed by Singer and Nicholson in 1972
electron transport chain required for the syn- (Figure 22.1). This proposes that the phospho-
thesis of ATP, and some are enzymes. In the lipid bilayer is fluid and that the phospholipid
specialized membranes of the rod cells in the molecules move in the lateral plane of the
retina, the principal protein is the light-sensi- membrane. However, there is very little
tive carotino-protein rhodopsin, which exchange of phospholipids between the two
accounts for approximately 90% of membrane halves of the lipid bilayer ('flip-flop' transi-
proteins. In the plasma membrane of the ery- tions) as this would involve the movement of
throcyte, a less specialized membrane, about the polar head groups of the phospholipid
20 different proteins have been isolated. A through the hydrophobic membrane core.
highly active membrane such as the inner One of the consequences of the minimal
mitochondrial membrane may contain hun- exchange of lipids between the two halves of
dreds of different proteins, some of which are the lipid bilayer is that it allows asymmetric
linked together to form complexes required distribution of phospholipids.
for specific biochemical processes. The fluidity of the lipid bilayer is influenced
Membrane proteins can be classified into by the type of lipid found in the membrane
two main groups: peripheral and integral pro- and, in particular, by the degree of unsatura-
Transport across membranes 315
Integral
~1""'t~Tt~,.,..~t=r+~ proteins
Lipid
bilayer
Figure 22.1 Structure of a lipid bilayer and the typical arrangement of peripheral and integral membrane
proteins.
tion of the fatty acids in membrane lipids. The of a tightly bound lipid fraction. These pro-
presence of a cis double bond introduces a teins are able to move in lateral plane of the
kink into fatty acids, thus the presence of membrane but are accompanied by their lipid
PUFAs with cis double bonds limits the extent coating. Some membrane proteins are clus-
to which the phospholipids in the bilayer can tered together to form large, functional units
pack together. This increases the fluidity of the such as receptors or transport complexes.
membrane, and also has the effect of main- Whilst these functional units are able to move
taining the fluidity of the membrane as the within the membrane, the relative positions of
temperature falls. The regulation of the fatty individual proteins within the complex
acid composition of membranes is not well remain constant. As with membrane lipids,
understood, but it is clear that both diet and there is very little movement of proteins in the
changes in environmental temperature influ- vertical plane of the membrane. Some integral
ence the ratio of saturated to unsaturated fatty proteins are glycoproteins, containing carbo-
acids in the lipid fraction. The incorporation of hydrate side chains of variable length and
sterols and sterol esters also affects membrane degree of branching. In the plasma membrane,
fluidity. For example, cholesterol has a dual at least, these glycoproteins are almost always
effect. Its ring structure is rigid and tends to orientated so that the carbohydrate protrudes
decrease membrane fluidity, but its presence from the surface of the cell.
disrupts the packing of the phospholipids and
tends to increase fluidity at low temperatures.
22.3 TRANSPORT ACROSS MEMBRANES
Together these effects may be useful in main-
taining membrane fluidity over a wide range Membranes act as selective permeability barri-
of temperatures. ers. With the exception of some hydrophobic
Integral proteins appear to have a layer or molecules which may be able freely to cross
domain of lipid associated with them which the membrane, most important biomolecules
may be required to maintain their functional are unable to pass through membranes by sim-
integrity. Extraction of integral proteins from ple diffusion because they are too large, too
membranes usually results in the co-extraction polar, or both. However, it is clear that many
different types of biomolecules are able to narrow binding specificity. A well character-
cross membranes by a process called mem- ized example of a membrane carrier system is
brane-mediated transport in which the mem- that for the transport of glucose across the
brane proteins playa very important role. plasma membrane of the human erythrocyte,
the so-called glucose permease system. The
protein carrier exists in two different confor-
22.3.1 MEMBRANE TRANSPORT MECHANISMS
mations. In one conformation the binding site
A small number of important molecules can is available on the extracellular surface of the
pass through membranes by diffusion because membrane. Attachment of glucose to the carri-
they are relatively non-polar, e.g. oxygen, nitro- er-binding site initiates a conformational
gen and methane. Polar compounds can only change in the protein, resulting in a move-
pass through membranes by a protein-mediat- ment of the glucose-filled binding site to the
ed process known as facilitated transport. intracellular surface of the membrane where
In general, it is assumed that the proteins glucose is released (Figure 22.3). Because the
which aid the movement of molecules across erythrocyte metabolizes glucose, its intracellu-
the membrane are integral proteins which lar concentration is usually lower than the
span the lipid bilayer. A number of models extracellular concentration and thus transport
have been proposed to explain how proteins of glucose occurs from out to in. However,
facilitate transport. In some cases they can because the process is freely reversible, glu-
form water-filled pores or channels through cose cannot be transported into the erythro-
the lipid bilayer which allow only those mole- cyte if the concentration in the surrounding
cules or ions small enough to fit into the chan- medium falls below that inside the cell.
nel to migrate from one side to the other. The carrier exhibits optimum activity
While the channel is open, bi-directional towards glucose. The transport constant, Kt,
movement of molecules or ions can occur by (analogous to the Michaelis constant, Km , for
diffusion (Figure 22.2). The net flux of a mole- an enzyme) for glucose is 1.5 mM. Other hex-
cule through the channel is determined by its ose sugars such as mannose and galactose
relative concentration on either side of the (which differ from glucose only in the position
membrane, with movement occurring from of one hydroxyl group) can also be carried, but
the area of high concentration to the area of the transport constants for these sugars are
low concentration. If the channel remains much higher, 20 and 30 mM respectively.
open the concentration of any particular mol- The glucose permease system is an example
ecule will reach equilibrium across the mem- of carrier proteins that transport single mole-
brane when transport continues to occur at cules, sometimes referred to as uniport sys-
equal rates in both directions. It is unlikely that tems. There are also examples of membrane
membrane channels remain permanently carriers which transport two different molecu-
open, as this does not allow for any control of lar species, a process known as co-transport. In
the transport process. The conformation of some cases the different molecules are trans-
channel proteins can be changed to close the ported in the same direction, a symport, but
channel, either directly by covalent modifica- they may also be transported in opposite
tion of the protein, e.g. phosphorylation, or directions, an antiport. The latter is used by
indirectly through the concerted effects of the erythrocyte membrane in the transport of
adjacent membrane proteins. waste CO 2 from the tissues to the lungs where
Membrane proteins may also act as carriers it is exhaled. The CO 2 is transported in the
within the membrane. These have binding blood plasma in the form of bicarbonate ions,
sites for molecules undergoing transport and, HCO:;. In the peripheral circulation, CO 2
by analogy with the active site of an enzyme, enters the erythrocyte and is converted to
the carrier-binding sites can exhibit broad or bicarbonate by carbonic anhydrase. The HCO:;
••
• .../
••••
• ••
Figure 22.2 Movement of molecules through protein channels in membranes.
ions are rapidly transported out of the ery- active transport, the pump is fuelled by the
throcyte in exchange for chloride ions (Cl-), an expenditure of energy provided by the
example of an antiport. Once in the lungs the hydrolysis of ATP, oxidation reactions or the
process is reversed, HCO; is taken up by the co-movement of a counter molecule down an
erythrocyte and Cl- ions are released. The electrical or chemical gradient using a sym-
HCO; is converted to CO 2, released into the port or an antiport.
plasma and expired (Figure 22.4). A particularly well understood example of
The channel- and carrier-mediated trans- ATP-driven active transport is the Na+, K+-
port processes described so far have all ATPase transport system found in all cell plas-
involved the movement of molecules from an ma membranes. There are large differences in
area of low concentration to an area of high the Na+ and K+ concentrations of intra- and
concentration, a process known as passive extracellular fluid (see Table 22.1).
transport. However, not all molecules cross The natural chemical gradients across the
membranes down a concentration gradient. plasma membrane tend to force K+ out of the
Movement of molecules from an area of low cell and Na+ into it. Cells maintain the differ-
concentration to an area of high concentra- ential concentration of Na+ and K+ across the
tion (i.e. against a concentration gradient) is plasma membrane by use of the Na+, K+-
called active transport. This movement can ATPase pump. The pump is an antiport which
only be achieved by the expenditure of ener- transports two K+ ions into the cell in
gy. A parallel can be drawn between passive exchange for every three Na+ ions carried out.
and active transport and the movement of In the process, one molecule of ATP is hydrol-
water between two tanks at different heights. ysed to ADP.
If the tanks are connected by a pipe, the The mechanism of the Na+, K+-ATPase is
hydrostatic pressure will cause the water to shown in Figure 22.5. The ATPase consists of
flow from the higher tank to the lower tank. two 0: and two 13 subunits. The 0: subunit con-
This can be compared with passive transport tains the ATPase activity and the Na+ and K+
where a concentration gradient acts as the binding sites. The function of the 13 subunit is
driving force. In order to move water from not known. The 0: subunit binds to three Na+
the low tank to the high tank a pump is need- ions on the intracellular surface of the plasma
ed which requires a supply of energy. In membrane. The ATPase then catalyses the
DO
o 0 o DO 0
DO 0 DO 0
o 0 o 0
1 Glucose Permease j
o DO 0 o 00 0
00 0 00 0
o 0 o 0
o
Figure 22.3 Mechanism of glucose permease.
transfer of the terminal phosphate group of revert to its original conformation in which K+
ATP to the a subunit which undergoes a con- binding sites are exposed on the intracellular
formational change, exposing the Na+ binding surface of the membrane in a low-affinity
sites on the extracellular surface of the mem- form, resulting in the release of K+ into the
brane and simultaneously reducing its affinity cell. The Na+ binding sites are now in the high
for Na+ so that the Na+ ions are released. At affinity form on the intracellular side of the
the same time, high-affinity K+ binding sites, membrane, bind to a further three Na+ ions,
previously inaccessible to the extracellular and the cycle is repeated.
environment, become available and bind two Maintenance of the correct intracellular
K+ ions. The a subunit is subsequently Na+ and K+ concentrations is vital for cell
dephosphorylated, allowing the carrier to function and it has been estimated that 25% of
Bi<:aibonate is released Tissues

into the blood stream cr
CO2 + H20 - HC03- + H+

Carbonic
anhydrase
Bicarbonate taken up Lungs

from the blood stream cr
CO2 + H20 _ HC03- + H+

Carbonic
anhydrase
CO2 released
into blood stream
and exhaled
Figure 22.4 Uptake and release of bicarbonate by erythrocytes in exchange for chloride ions, part of the
mechanism of CO 2 removal from tissues.
energy expenditure in resting humans can be ures suggest that approximately 50 kg of ATP
attributed to the Na+, K+-ATPase. This is hydrolysed in a 24-hour period in order to
accounts for the consumption of very large maintain Na+ and K+ concentrations in the
quantities of ATP: during a 24-hour period the cell.
energy requirements of an adult human Active transport can also be fuelled by the
account for the hydrolysis of about 200 kg utilization of electrochemical gradients across
ATP, although at anyone time the total membranes. This can be illustrated by refer-
amount of ATP in the body is about 50 g. ence to the transport of lactose into the bacteri-
There is, therefore, continual resynthesis of um Escherichia coli. The plasma membrane of E.
ATP from ADP and Pi" Nonetheless, these fig- coli contains an electron transport chain which
Table 22.1 Concentration of sodium and potassium tons and lactose) via a symport (Figure 22.6).
in intracellular and extracellular fluid in mammalian Proton gradients are not only used to fuel
cells active transport but, as discussed in Chapter
12, they can also be used to provide the energy
Ion Intracellular Extracellular
concentration concentration needed to synthesize ATP in specialized mem-
(mM) (mM) branes such as the inner mitochondrial mem-
brane. These proton-dependent processes are
5-15 145-150 examples of chemiosmotic coupling.
140-145 5-10 In the membrane transport described so far,
the molecule undergoing transport across the
acts as a proton pump, transporting protons membrane is not modified during the trans-
from the intracellular compartment to the sur- port process. There is another form of trans-
rounding medium. The proton gradient creat- port in which the transported molecule is
ed by this mechanism is an electrochemical chemically modified before release from the
gradient referred to as a proton motive force membrane. This process is known as group
(PMF).The plasma membrane is impermeable translocation and is typified by the phospho-
to protons, and the PMF is coupled to a trans- transferase system (PTS) for sugars, which has
port protein in the membrane which trans- been well characterized in several bacteria,
ports both lactose and protons into the cell. By particularly E. coli, Salmonella typhimurium and
this process, protons return to the cytoplasm Staphylococcus aureus (Figure 22.7). The trans-
down a concentration gradient and provide port complex consists of three enzymes and
the energy required to drive the lactose into one non-enzymic protein. Enzyme I and HPr
the cell against a concentration gradient of are soluble proteins located in the cytoplasm;
1:100. This is an example of co-transport (pro- enzyme II is an integral protein located in the
Figure 22.5 Mechanism of Na+, K+-ATPase.

I Extracellular I I Intracellular I
Figure 22.6 Uptake of lactose in Escherichia coli by a proton/lactose symport.
cell membrane which acts both as a permease complexes which interact with hormones and
and catalyses the final transfer of phosphate other bioactive molecules. Binding of a hor-
from phosphoenolpyruvate (PEP) to the trans- mone to its receptor can have a number of
ported sugar. The latter step also requires the consequences. Hormones such as the cate-
presence of enzyme III which is associated cholamines (adrenalin and noradrenalin)
with enzyme II in the membrane. interact with plasma membrane receptors and
The specificity for the sugar to be transport- activate the enzyme adenyl cyclase via a
ed is associated with enzymes II and III. PTS receptor-associated G-protein (GTP-binding
can be considered a special form of active protein) mechanism. The primary effect of
transport in that it utilizes PEP as a high-ener- these interactions is to increase the intracellu-
gy phosphate donor. However, it differs from lar concentration of 3',5', cyclic AMP (cAMP),
the Na+, K+-ATPase previously described one of the so-called 'second messengers',
because the chemical modification of the which initiates further metabolic changes in
transported sugar means that there is no direct the cell by activation of cAMP-dependent pro-
accumulation of sugars against a concentra- tein kinases. The activation of triacylglycerol
tion gradient. lipase (hormone-sensitive lipase) and the reg-
ulation of glycogen metabolism are brought
about by such cAMP-mediated mechanisms.
22.3.2 OTHER FUNCTIONS OF MEMBRANES
Other hormones, such as insulin, interact with
In addition to their role in transport processes, receptors which have an intracellular tyrosine
membranes have numerous other important kinase. This catalyses phosphorylation of tyro-
functions. They are involved in the process of sine residues on target proteins which then
signal transduction. Most cellular membranes, initiate changes in cellular metabolism. These
but particularly the plasma membrane, con- mechanisms are described in more detail in
tain receptors. These take the form of protein Chapter 30.
, JEnzyme III
. . J<y¥+-----I Enzyme I I
PEP
X ' Pryuvate
Figure 22.7 Group translocation by the phosphotransferase system.
Proteins on the extracellular surface of plas- oxidative phosphorylation. TCA-cycle interme-

ma membranes are involved in the cell-to-cell diates can also be used as the starting point for
contacts and cellular recognition. These pro- the synthesis of amino acids for protein syn-
teins are often glycoproteins. In animals, many thesis, of sugars for polysaccharide synthesis
immune response mechanisms are triggered and of fatty acids for lipid synthesis. These
by the presence of organisms which contain processes do not occur at random but are coor-
'foreign' cell-surface proteins. This initiates the dinated. In many cases they are influenced by
activation of the immune system and the pro- the supply of nutrients in the extracellular
duction of antibodies, leading to the destruc- environment and, in complex multicellular
tion of the foreign organisms. organisms, the relative importance of one
metabolic pathway over another is regulated
with respect to the stage of growth, reproduc-
22.4 PRINCIPLES OF METABOLIC
tive state and tissue specialization.
REGULATION
There are three main ways in which metab-
Part Two describes the metabolism of the main olism is coordinated through the regulation of
constituents of the cell, carbohydrates, proteins enzymes. Firstly, by allosteric regulation,
and lipids, and the ways in which their break- through changes in the concentration of spe-
down can provide energy and substrates for cific enzyme activators and inhibitors.
biosynthetic processes. For example, sugars Secondly, by covalent modification of an
and fatty acids can be broken down to supply enzyme to bring about a change in its activity.
acetyl-CoA which can be oxidized in the TCA Thirdly, by changing the amount of enzyme
cycle. The reduced cofactors generated (NADH present through modification of gene expres-
and FADHz) can be used to synthesize ATP by sion and protein turnover.
Principles of metabolic regulation 323
22.4.1 ALLOSTERIC REGULATION are often cofactors, such as ATP, NADH and
NADPH, the concentration of which reflect
Most metabolic processes involve a series of
energy status or redox potential in a cell. In
biochemical transformations in which a sub-
addition, the end product of a pathway or an
strate is converted to an end product via a
intermediate downstream of the allosteric
number of discrete steps, each step being
enzyme may act as a regulator. In this latter
catalysed by a specific enzyme. This sequence
case the activity of the allosteric enzyme is
of reactions constitutes a metabolic pathway.
usually inhibited, and this type of regulation is
Regulation of the flow of metabolites through
referred to as feedback or end-product inhibi-
a metabolic pathway occurs by modifying the
activity or amount of one or more of the tion. This type of mechanism allows the cell to
'sense' when a particular end product is in
enzymes catalysing the individual steps. In a
excess of the requirement. Feedback inhibition
relatively simple pathway this regulation may
slows down the reaction catalysed by the rate-
be achieved via only one enzyme, usually the
limiti~g enzyme of the pathway, thereby
one which catalyses the first reaction in the
reduc10g the rate of production of the end
pathway. This is referred to as the committed
product. If the end product is used by other
step in a metabolic pathway. In more complex
metabolic processes, its concentration in the
pathways, which have many branch points
cell will fall and the inhibition of the rate-lim-
and the potential to convert the substrate to a
iting enzyme is relieved, allowing more end
number of end products, the first enzyme after
product to be formed. This type of mechanism
each branch point is usually regulatory (Figure
allows acute control of metabolic pathways.
22.8). These allosteric enzymes are often com-
The biosynthetic pathways of many amino
plex and are controlled by factors other than
acids are branched and are regulated by feed-
the availability of substrate. They are usually
back inhibition.
rate-limiting enzymes which control the flux
of metabolites along a specific pathway. Other
~nzymes in the pathway are normally present 22.4.2 COVALENT MODIFICATION
10 excess and their activity is determined
largely by substrate supply, which in turn Th~s . type of modification may alter enzyme
actIvity by changing the conformation of the
depen?s on the activity of the enzyme
protein which affects the shape and size of the
catalys10g the preceding reaction.
A number of features are typical of allosteric active site. The most common type of covalent
mod~fication is phosphorylation or dephospho-
enzymes (see also Chapter 6). They are regu-
lated by the presence of specific signal mole- rylation of one or more amino acids, most often
cules which are distinct from the substrate and serine residues. The control of glycogen metab-
olism provides a good example of this type of
I
S-T-U-V enzyme regulation. Glycogen breakdown is
Es Es E7 • catalysed by the enzyme glycogen phosphory-
E1 E2 E3 E4 +----------' lase which exists in two forms, glycogen phos-
\ :\E
A*S-C-D phorylases a and b. Phosphorylase a is the most
active form of the enzyme; phosphorylase b is
a + - - - - - - - - --
"
... _ , .. " Eg El0 Ell'I
relatively inactive. The enzyme consists of two
--- W-X-Y-Z subunits, each of which contains a specific ser-
Figure 22.8 Feedback inhibition of allosteric ine residue which undergoes phosphorylation,
enzymes in a branched metabolic pathway. resulting in the conversion of phosphorylase b
(E1-Ell represent the enzymes catalysing individ- to phosphorylase a. In most cases of covalent
ual steps; A-D, S-V and W-Z represent the sub- modification by phosphorylation the phos-
strates and products of the reactions.) phate donor is ATP and the phosphorylation
reaction is catalysed by a protein kinase, in this The polypeptide hormone glucagon acts main-
case called phosphorylase kinase. This kinase is lyon liver glycogen metabolism through acti-
itself activated by a non-specific protein kinase. vation of adenyl cyclase. Its effects are similar to
Conversion of phosphorylase a back to phos- adrenalin in that it increases the supply of glu-
phorylase b occurs by removal of the phosphate cose-6-phosphate. However, as liver has an
groups catalysed by another enzyme, phospho- active glucose-6-phosphatase, there are two
rylase phosphatase. major fates for the glucose-6-phosphate: either
Phosphorylation/dephosphorylation is also conversion to glucose and secretion into the
used to control the activity of glycogen syn- blood stream, or entry into the glycolytic path-
thesis through the enzyme glycogen syn- way.
thetase, which also exists in two forms, a and b. Often allosteric regulation and covalent
The active form, glycogen synthetase a, is the modification act in concert on the same
dephosphorylated enzyme. It is inactivated by enzyme. Glycogen phosphorylase is one such
phosphorylation, catalysed by protein kinase, enzyme. In addition to the covalent modifica-
and reactivated by removal of phosphate tion described above, the activity of this
groups by phosphoprotein phosphatase. Thus enzyme is regulated by the intracellular con-
the regulation of glycogen metabolism is centrations of ATP, AMP and glucose, all of
brought about by reciprocal changes in the which act as allosteric effectors. The enzyme
activities of glycogen synthetase and glycogen is strongly inhibited by ATP and glucose and
phosphorylase. Phosphorylation activates is activated by AMP. Thus, the activity of the
glycogen phosphorylase and inhibits glycogen enzyme is sensitive to the energy status of the
synthetase, whereas dephosphorylation has cell. When there are adequate supplies of
the opposite effect (Figure 22.9). energy in the form of glucose and ATP, the
The reciprocal regulation of these enzymes cell has no need to degrade glycogen to glu-
eliminates the possibility of a futile cycle involv- cose. However, high levels of AMP, which
ing breakdown of glycogen to produce glucose- reflect reduced availability of ATP, stimulate
I-phosphate and its reincorporation into glyco- release of glucose from glucogen and increase
gen. In fact, in animals, the control of glycogen the supply of glycolytic substrate for ATP
metabolism is linked to whole-body metabolic synthesis.
needs via fluctuations in the levels of circulat-
ing adrenalin and glucagon. Adrenalin acts pri-
22.4.3 CHANGES IN THE AMOUNT OF ENZYME
marily on glycogen metabolism in muscle. It is
secreted in response to nutritional and physio- A third way in which regulation is achieved is
logical stress, situations in which glucose sup- by controlling the synthesis of enzymes.
ply to muscle tissue may become limiting. It Whilst allosteric regulation and covalent mod-
acts on cell membrane adrenergic receptors to ification can be considered acute responses to
increase the intracellular concentration of changes in metabolic state, control of the rate
cAMP. The net effect of this increase is the inhi- of synthesis of enzymes is a longer term effect.
bition of glycogen synthetase, activation of It involves the regulation of gene expression
glycogen phosphorylase and an increase in the and is discussed in more detail in Chapter 21.
supply of glucose-I-phosphate. Glucose-I- This type of regulation is typified by the lac
phosphate is converted to glucose-6-phosphate operon in bacteria which controls the produc-
by phosphoglucomutase and, as muscle cannot tion of J3-galactosidase in response to nutrient
release free glucose due to a lack of glucose-6- supply. Repression of enzymes required for
phosphatase, the glucose-6-phosphate enters amino acid synthesis in bacteria is controlled
the glycolytic pathway and is used to produce in a similar manner. Changes in the amount of
the ATP needed to power muscle contraction. enzyme also occur in plants and animals as a
AdrenaJin/Glucagon
Receptor:::.1'\!! me~~~~ne
.-
Adenyl cyclase
~ Glycogen
/ ~ synthetase a~
Inactive
protein kinase
Active
protei" kinase
...• fl
'-
~ Phosphoprotein
~ . .. phosphatase
: Glycogen
~ synthetase b i
Inactive Active
phosphorylase phosphorylase
kinase kinase
~Glycogen
Glycogen
phosphorylase a phosphorylase b-®
pX
I Phosphorylase
phosphatase
Figure 22.9 Control of glycogen metabolism by covalent modification initiated by adrenalin and
glucagon.
regulatory mechanism. However, the ways in in these pathways are illustrated in Figure
which this is brought about differ between 22.10.
prokaryotes and eukaryotes. The main function of glycogen stored, for
example, in muscle tissue is to provide glu-
cose-6-phosphate for ATP production. In vivo,
22.4.4 COORDINATED REGULATION OF
in tightly coupled mitochondria, the flux of
PATHWAYS
electrons through the electron transport chain
The direction of metabolism is controlled by and the synthesis of ATP are regulated by the
the coordinated regulation of interconnecting energy requirements of the cell. Utilization of
pathways. This regulation is brought about ATP for metabolic processes is the trigger
both by allosteric regulation of enzyme activi- which initiates oxidative metabolism. The
ty and by covalent modification of enzymes in energy status of the cell is reflected in the ratio
response to changes in hormonal status. This of [ATP]/([ADPJ + [PJ}. This ratio is normally
can be illustrated by examining the way in high, i.e. high cellular concentrations of ATP
which glycogen metabolism, glycolysis, the and low cellular concentrations of ADP. Under
TeA cycle and oxidative phosphorylation are these conditions, the supply of ADP for ATP
coordinated. The allosteric effects of substrate synthesis is limiting and the flux of protons
and coenzyme concentrations on the enzymes through the FaFl-ATPase is low. The energy
It =~~
Glycogen
highATP, g/lIco$&"
Glucose-1-phosphate
Glucose • +
Glucose-6-phosphate
high~+ ~
highATP,OItrafe +~t high ADP
Fructose-1,6-bisphosphate
1
pho8ph!noIPyrUvate
.~~fI'" th(liADP
Pyruvate
+ ~ ·t.~~
Acetyl-CoA
Citrate
+
¥_A::;_am 1 ADP+~
Isocitrate
1..--. t~11
ATP
.~,ATP+ '>.. <7 ,~ HzO

$.. ucaI~_COA
Malate
,.~ t ~
--+----Oxaloacetate
Figure 22.10 A summary of glycolysis, the TCA cycle and electron transport showing the main points
where the metabolism of glucose and glycogen is regulated by char,ges in the concentration of interme-
diates: NAD+, NADH, ATP and ADP.
required for the electron transport chain to proton gradient is high, electron transport
pump electrons across the inner mitochondri- slows down and the rate at which NADH and
al membrane increases as the magnitude of FADHz are reoxidized decreases. This leads to
the proton gradient increases; thus when the a more reduced environment in the cell
because of the high NADH/NAD+ ratio. The and amino acid synthesis. In contrast, when
combined effect of a high ATP concentration blood glucose falls the secretion of insulin
and the high NADH/NAD+ ratio reduces the decreases and that of glucagon increases.
rate of oxidative metabolism. This is brought Glucagon not only stimulates the mobiliza-
about by: tion of glycogen reserves (section 22.4.2) but
also stimulates gluconeogenesis and inhibits
• the direct effect of ATP on key regulatory
glycolysis. The balance between these two
enzymes in the TCA cycle, glycolysis and
pathways is controlled by a relatively newly
glycogenolysis, e.g. a-ketoglutarate dehy-
discovered compound, fructose-2,6-bisphos-
drogenase, citrate synthase, pyruvate dehy-
ph ate (F-2,6-P). This is synthesized by a sec-
drogenase, pyruvate kinase, phosphofruc-
ond type of phosphofructokinase referred to
tokinase-I and glycogen phosphorylase;
as PFK2 (to distinguish it from PFKI which
• the lack of NAD+ and FAD which reduces
catalyses the mainstream reaction of glycoly-
the activity of dehydrogenases of the TCA
sis). F-2,6-P is broken down to fructose-6-phos-
cycle and glycolysis;
ph ate (F-6-P) by the action of fructose-2,6-bis-
• the consequent build-up in concentration
phosphatase. The unique feature of the metab-
of intermediates which cause feedback
olism of F-2,6-P is that the enzymes responsi-
inhibition of the enzymes which produce
ble for its synthesis and degradation are locat-
them, e.g. succinate inhibits a-ketoglu-
ed on the same protein, which acts as a bifunc-
tarate dehydrogenase, citrate inhibits cit-
tional enzyme. The nature of the enzyme
rate synthase, acetyl-CoA inhibits pyruvate
activity expressed by this protein is deter-
dehydrogenase, and glucose-6-phosphate
mined by its phosphorylation state. In the
inhibits hexokinase.
dephosphorylated state it functions as PFK2,
This concerted regulation ensures that but when phosphorylated it acts as F-2,6-bis-
glycogen and glucose are not wastefully phosphatase. F-2,6-P is synthesized from F-6-P
metabolized when the energy requirements of and is a potent activator of PFKl. Thus when
the cell are low. Depletion of ATP reserves the F-6-P concentration is high, F-2,6-P is pro-
rapidly releases the ATP inhibition of oxida- duced, PFKI activity is increased, and the flux
tive metabolism, increases the rate of electron of glucose through the glycolytic pathway
transport and reduces the NADH/NAD+ ratio increases. The primary regulator of PFK2 and
in the cell, allowing oxidative metabolism to F-2,6-bisphosphatase activity is glucagon,
increase until a new energy equilibrium is which acts via cAMP and protein kinase to
established. inhibit PFK2 and activate F-2,6-bisphos-
Superimposed on this level of control is a phatase. The net effect of glucagon is to reduce
further tier of regulation, the covalent modifi- the concentration of F-2,6-P and shift the bal-
cation of a number of key enzymes. This type ance between glycolysis and gluconeogenesis
of regulation modifies the metabolic activity of in favour of the latter. Thus, in liver, glucagon
cells and tissues in response to changes in promotes the production of glucose by stimu-
whole body requirements. The control of car- lating both glycogenolysis and gluconeogene-
bohydrate metabolism via the pathways con- sis. Glucagon also inhibits glycolysis via pyru-
sidered above again provides good examples. vate kinase. The activity of this enzyme is
In animals, the availability of food is reflected reduced by phosphorylation initiated by
in changes in the concentration of glucose in glucagon and mediated via a cAMP depen-
the blood. The endocrine response to an dent kinase (Figure 22.11).
increase in blood glucose levels is secretion of Allosteric regulation and covalent modifica-
insulin from the pancreas, which stimulates tis- tion are found in other areas of metabolism.
sue uptake of glucose for glycogen, fatty acid For example, in fatty acid metabolism the pres-
Glucose glucagon
J1 1
l-:~~-~T
'----f--+------'~ ~/: ill~/
-ve
F-1,6-bisP . . - - - -" +ve
/
=
t~~~1
Pyruvate
Figure 22.11 The effect of glucagon on glycolysis and gluconeogenesis.
ence of citrate, an intracellular indicator of this is prevented in several ways. Firstly, a fall
high energy status, stimulates acetyl-CoA car- in blood glucose stimulates lipolysis. The pres-
boxylase which catalyses the production of ence of high levels of acyl-CoAs in tissues
malonyl-CoA, a substrate for fatty acid syn- where fatty acid synthesis takes place inhibits
thetase. This has the effect of channelling acetyl-CoA carboxylase and limits substrate
excess energy to fat storage. Insulin, which availability for the synthesis of fatty acids.
acts as a sensor of high blood glucose levels in Secondly, glucagon and adrenalin concentra-
animals, promotes the activation of pyruvate tions increase in response to reduced energy
dehydrogenase and ATP-citrate lyase via a supply. Both of these hormones cause deacti-
phosphorylation mechanism. Both of these vation of acetyl-CoA carboxylase via a phos-
enzymes catalyse reactions leading to the pro- phorylation mechanism. These relationships
duction of acetyl-CoA which can be used for are summarized in Figure 22.12.
fatty acid synthesis. To ensure that newly syn-
thesized fatty acids are not immediately oxi-
22.4.5 COORDINATION OF METABOLISM IN
dized, malonyl-CoA inhibits carnitine acyl-
DIFFERENT TISSUES
transferase I, thus preventing fatty acids from
being transported into the mitochondria In mammals, where tissues have developed
where [3-oxidation takes place. with specialized biochemical functions, the
When energy supply is limiting it would be endocrine system serves as a means of inte-
wasteful to convert glucose to fatty acids, and grating tissue metabolic activity through
Insulin
"
Citrate
I
~ .. - - - - -
.. ..
Acetyl-CoA
caIbox'
ActItyI-J;oA
.. - - - - - - - - - - - Glucagon
--,
\
I
I
,, - - - - - Malonyl-CoA
I
----
Fattyacyl-carnitine
Figure 22.12 The control of lipid metabolism is coordinated through allosteric regulation and covalent
modification of enzymes.
changes in hormonal status. This can be illus- such animals are in a state of negative energy
trated by considering the relationship balance. The drain on blood glucose causes a
between carbohydrate and lipid metabolism in decrease in the plasma concentration of
liver and adipose tissue, and the way in which insulin and a corresponding increase in that of
particular physiological states, such as preg- glucagon. Plasma catecholamine levels also
nancy and lactation in sheep and cows, increase. These hormonal changes modify the
change the balance and direction of their metabolic activity of liver and adipose tissue.
metabolic activity. The increase in glucagon levels stimulates
A well nourished animal fed above its main- hepatic gluconeogenesis and channels all
tenance level is in positive energy balance, and available glucogenic substrates towards the
therefore glucose synthesis in the liver is ade- synthesis of glucose. At the same time, the hor-
quate to meet the needs of growth and allow monal shift has profound effects on adipose
synthesis of glycogen and deposition of fat. In tissue metabolism (Figure 30.11). Firstly, the
contrast, in late pregnancy in sheep carrying decrease in plasma insulin concentration
multiple foetuses, or in early lactation in the reduces the uptake of glucose by adipocytes
high-yielding dairy cow, the glucose demand and inhibits fatty acid synthesis. This limits the
for the foetuses or the mammary gland is supply of substrates (glycerol-3-phosphate
exceptionally high and feed intake cannot and fatty acids) for triacylglycerol synthesis
match metabolic requirements. As a result and reduces lipogenesis. Secondly, the
i
amino
acids
phosphoenolpyruvate
f lactate
I fatty acids I
,::/~
.. ~.
.. " .
l:;a~
/3-oxidation
Phosphoenolpyruvate
carboxykinase
'acetyl-CoA
,..
Ketone
bodies
amino ~(xaloacetate
acids -4 ~C02
J-co,
TCA Cycle -ketOQI:tarate' .. .... am.ino
a ~ aCids
\
~ fum~te SUCCinYI-<?~A
1
.
ammo -4' ""- ./ ~.
acids Succinate 4t' ~
amino
acids
Propionate
Figure 22.13 Negative energy balance in animals leads to the production of ketone bodies. The solid
arrows indicate the direction of flow through metabolic pathways.
increase in glucagon and catecholamine con- These fatty acids can be utilized for energy
centrations stimulates the activity of triacyl- production by some other tissues in the body
glycerol lipase and thus increases lipolysis. In and therefore have a sparing effect on glucose
addition, these hormones inhibit fatty acid utilization. Liver is an important site of fatty
synthesis. This lipolytic effect may be further acid oxidation which produces acetyl-CoA.
enhanced by release of the inhibitory effect of Under normal circumstances this would be
insulin on lipolysis. The net effect of these further oxidized in the TCA cycle after its con-
changes in adipose tissue is the mobilization of densation with oxaloacetate to form citrate.
fatty acids. However, during negative energy balance,
BLOOD LIVER
ADIPOSE TISSUE
,
NEFA ~Acety~COA
r
~---"'·~Glycerol
OxAC-,
\
+ NEFA
I
I PEP . . . .
PREDOMINANT
,~
•
Citrate
PATHWAYS
t· ;
TGs
FATTY ACYL-CoA OF KETOSIS
I
I I Glucose \ ~ _.. _ ' I
\ I
~ - - G-3-P ... - '
~ ~ - Glucose" ~ jl-OH Butyrate.....I-_ _ _•
Acetoacetate
MAMMARY GLAND
OR
FOETUS
Figure 22.14 The relationship between metabolism in adipose tissue and liver during ketosis.
oxaloacetate is in short supply as any that is subclinical form of ketosis. When the lambs
produced (for example from propionate or are born or the level of lactation falls, these
amino acids) is rapidly syphoned off for the animals will eventually return to positive
synthesis of glucose. In this situation, acetyl- energy balance. When this occurs the require-
CoA cannot be oxidized and is instead con- ment for hepatic gluconeogenesis is reduced
verted to ketone bodies (Figure 22.13). These and, in adipose tissue, lipogenesis takes over
can be exported from the liver to other tissues from lipolysis. The relationship between
which use them as substrates for energy pro- metabolism in adipose tissue and liver during
duction. The synthesis of ketone bodies is ketosis is summarized in Figure 22.14. The
described in Chapter 13. changes described above are an extreme case
The presence of excessive concentrations of of a normal metabolic response to changes in
ketone bodies in the blood results in the devel- energy intake. Less dramatic shifts in the
opment of the clinical condition known as metabolism of liver and adipose tissue occur
ketosis. The two major ketone bodies are acids continually in animals and humans. For exam-
(l3-hydroxybutyric acid and acetoacetic acid), ple, short periods of fasting, even the
which, if present in excess in the blood, can overnight fast between an evening meal and
cause acidiosis (a decrease in blood pH), which breakfast, will cause an increase in glycogenol-
in some cases is fatal. ysis, gluconeogenesis and lipolysis.
It is not uncommon for the pregnant ewe
and lactating cow to suffer from a less severe,
PART THREE
STRATEGIES FOR PROCESSING OF NUTRIENTS

IN PLANTS
SEEDS AND GERMINATION 23
23.1 Seeds and plant development 335
23.2 Seeds as food and agricultural 335
commodities
23.3 Seed composition 336
23.3.1 Seed carbohydrates 336
23.3.2 Seed lipids 337
23.3.3 Seed proteins 337
23.3.4 Seed minerals 339
23.4 Germination 340
23.4.1 Starch breakdown 340
23.4.2 Beer and whisky production 341
23.4.3 Protein breakdown 342
23.4.4 Lipid breakdown 342
23.1 SEEDS AND PLANT DEVELOPMENT tance are cereals, legumes and oilseeds. The
cereal staples (wheat, rice and maize) togeth-
The seed is the means by which a plant repro-
er provide more than 50% of the world's food
duces and multiplies, and which allows it to
persist through adverse conditions. Inside a supply, and a further 13% is provided by
legume seeds (peas, chickpeas, beans, lentils,
protective seed coat, it consists of an embryo
soyabeans and cowpeas). Cereal seeds are
together with nutrient reserves, and which sup-
particularly important because, in addition to
port the growth of the embryo until the seedling
supplying energy, they are the major global
IS able to carry out photosynthesis and obtain
nutrients from the soil via the root system. source of protein in human and animal diets.
Most cereal proteins are deficient in some
During the phase of seed development and
essential amino acids. Where they provide
maturation on the mother plant, the embryo
most of the protein intake, it is likely that the
develops from the fertilized ovule and the
diet as a whole will be deficient in these
nutrient reserves may accumulate in either the
amino acids. Thus attempts have been made
endosperm or the cotyledons, which form part
to improve the quality of cereal proteins as
of the embryo itself.
this would improve human nutrition, partic-
Wherever the nutrients are stored, their
ularly in developing countries where they are
function is to provide the materials which are
the major source of nitrogen in the diet. This
needed to support the growth of the develop-
could reduce the need for high-protein addi-
ing seedling. They must thus act as a source of
tives such as soyabean, fish or pure amino
energy, carbon, nitrogen, sulphur and other
acids in the nutrition of man or other mono-
major and trace elements.
gastric animals.
In addition to nutritional uses, seeds also
23.2 SEEDS AS FOOD AND AGRICULTURAL have important commercial and industrial
COMMODITIES applications. Thus, cereal grains may be malt-
Seeds are major world food crops. The princi- ed to provide raw materials for the brewing
pal seeds which are of agricultural impor- and distilling industries, and starch and oils
336 Seeds and germination
may be extracted from seeds and processed to grown principally as a source of oil for the
provide important industrial raw materials. food industry. They contain very little carbo-
hydrate, but the meal which remains after
extraction of the fat is a source of good quality
23.3 SEED COMPOSITION
protein which can be used in animal feeds.
Although seeds of different species have the
same functions, their composition varies a
great deal. Typical compositions of some dif- 23.3.1 SEED CARBOHYDRATES
ferent types of seeds are shown in Table 23.1. Seed carbohydrates consist principally of
In many seeds the energy reserves consist of starch, which is found as granules in the
carbohydrate, principally starch. In others endosperm. Starch consists of a mixture of
starch may be almost completely absent and amylose (straight-chain structure) and amy-
instead large amounts of lipid may be present. lopectin (branched-chain structure) and the
Because of its higher energy content, the use proportions of these two forms are largely
of lipid, instead of starch, as an energy store genetically controlled. In barley, wheat, oats,
allows seeds to be smaller, lighter and more maize and rye there is between 27 and 29%
easily dispersed (Chapter 9). amylose in the starch whereas in rice there can
Cereal grains contain large amounts of
be up to 37% amylose. In some mutants the
starch. Protein levels are moderate but, as dis-
proportions may be quite different, e.g. in
cussed elsewhere, the quality is poor for feed-
'waxy' maize there is virtually no amylose. The
ing to non-ruminants. The lipid content is low,
proportion of amylose present in the starch
but it is higher in oats and maize than in most
increases during development of the grain. As
other cereals.
high-amylose starch has the ability to form
The pea is typical of many legumes and
films and fibres, it has considerable industrial
contains less carbohydrate and more protein
potential. The composition affects the physical
than cereals. The protein is also of higher qual-
properties of the grain so that high-amylose
ity than cereal protein. There is very little fat in
starch leads to a 'floury' endosperm, whilst
most legumes but soyabeans are rather differ-
ent, containing moderate amounts of both fat low-amylose starch causes the endosperm to
and carbohydrate, and being intermediate in be 'flinty'. In granules the starch exists in an
composition between legumes and oilseeds organised, semi-crystalline form which is
such as rapeseed, linseed and sunflower. almost entirely due to the amylopectin frac-
In oilseeds the main storage reserve is in the tion. The amylose and amylopectin molecules
form of triacylglycerols, and these crops are are probably held together through the forma-
tion of hydrogen bonds and there is also some
Table 23.1 The approximate composition of vari- tightly bound water which is necessary for the
ous types of seeds ordered structure of the starch granule. The
crystalline structure of starch in the granules
Seed Water Carbohydrate Protein Fat has to be broken down before complete
(g kgfresh
wt- 1)
enzymic breakdown can take place. Physical
processing of starchy feeds, e.g. dry heat or
Wheat 140 660 132 20 hydrothermal processing, leads to gelatiniza-
Barley 106 836 79 17 tion of the starch and may have a substantial
Oats 89 728 124 87 effect on the nutritive value of feeds. Cereal
Dried peas 133 500 216 13 starch granules tend not to burst on gela-
Soyabean 70 235 368 235 tinization, whilst those of some other plants
Rapeseed 70 250 212 486
(e.g. potato tubers) do.
Seed composition 337
Starch from some sources is an important mainly enzymes needed in the developing or
industrial raw material. It may be phosphory- germinating seed. The storage proteins act as
lated which alters its physical properties and reserves of nitrogen for the germinating seed.
potential uses. Their properties, like their functions, are
rather different from most other proteins.
They have a relatively high nitrogen content
23.3.2 SEED LIPIDS
to allow them to store the nitrogen efficiently,
Plants in general do not contain large amounts they are stable to desiccation and rehydration,
of lipid: for example, the content in cereal and are readily broken down by proteolytic
grains varies from 10-30 g kg-I dry matter in enzymes during germination.
wheat, barley and rye to 40-60 g kgl dry mat- The NPN fraction makes up 2-12% of the
ter in oats and maize. This lipid is concentrat- total seed nitrogen. It consists principally of
ed in the embryo (rice embryos contain a par- free amino acids, nitrate, ammonia and small
ticularly high concentration: 350 g kgl dry peptides. Although it is quite a small propor-
matter within the small amount of embryo). tion of the total nitrogen under most circum-
The lipid is in the form of triacylglycerols and stances, it may rise as high as 35% when plants
is quite unsaturated, containing much linoleic are subjected to environmental stresses.
and oleic acid, and it therefore tends to oxidize
readily. The presence of these unsaturated
Cereal proteins
lipids in the diet may lead to the production of
unacceptably soft fat in pigs. The protein content of cereal grains is normal-
Some commercially important crops accu- ly between 80 and 120 g kgl dry matter, and it
mulate large amounts of oils in their seeds is present in highest concentrations in the
(Table 4.4). This is often accompanied by use- embryo and aleurone layer.
ful quantities of protein, enhancing the value The nutritional quality (amino acid compo-
of the crop still further. Important oilseed sition) of most cereal proteins is poor in com-
crops include sunflower, rapeseed, soyabean, parison with those from other sources (Table
castor bean and groundnut. 23.2). The general levels of essential amino
Chemically, plant seed oils are triacylglyc- acids are low, but the limiting amino acids
erols. These contain fatty acids of varying vary in different cereals. Thus lysine is the first
chain lengths (Table 4.3). A number of plant limiting amino acid in maize, sorghum, barley,
oils have important industrial uses, including wheat and triticale. The second to become lim-
manufacture of cooking fats, lubricants, soaps, iting in maize is tryptophan; in barley and
pharmaceuticals, fuels and other chemicals. sorghum it is threonine. Diets in which these
Some oilseeds have toxic constituents. For cereals predominate tend to be deficient in
example, rape and mustard both contain eru- these amino acids, unless the diet also contains
cic acid (Chapter 4) and glucosinolates protein from other sources in which they are
(Chapter 3). Because of the nutritional impor- more abundant.
tance of rapeseed, varieties with low levels of Cereal proteins are subdivided into albu-
both constituents have been developed. mins, globulins, prolamins and glutelins on
the basis of their solubility (Chapter 5).
Although this classification is rather crude, the
23.3.3 SEED PROTEINS
classes correlate to a considerable degree with
The nitrogen in most seeds is found as a pro- the function and amino acid composition of
tein and a non-protein nitrogen (NPN) frac- the proteins. In most cereals the storage pro-
tion. The proteins consist of specialized stor- teins are mainly prolamins or glutelins, whilst
age and non-storage proteins. The latter are non-storage proteins are albumins and globu-
Table 23.2 Essential amino acid content of cereals and other foods
Barley Rice Pea Cabbage Beef Cod

Amino acid (g amino acid kg food- 1)
He 3 2.6 2.5 1.0 10.4 9.2

leu 5.7 5.6 4.0 1.8 16.3 14.7
lys 2.2 2.5 4.3 1.0 18.5 17.0
met 1.4 1.4 0.6 0.3 5.5 5.0
phe 4.3 3.3 2.7 1.0 9.1 7.2
thr 2.8 2.3 2.3 1.2 9.4 8.3
trp 1.4 0.9 0.6 0.3 2.6 2.0
val 4.2 3.9 2.7 1.4 10.7 10.0
arg 4.1 5.1 5.4 2.8 13.7 11.1
his 1.8 1.6 1.3 0.9 7.5 5.0
Data from Amino Acids and Fatty Acids (1st Supplement to The Composition
of Foods, 4th edition) are reproduced with the permission of The Royal
Society of Chemistry and the Controller of Her Majesty's Stationery Office.
lins. As the distinction between albumins and determined mainly by the composition of the
globulins is not clear-cut, they are often prolamin fraction, as this is most abundant,
grouped together. In most cereals there is also accounting for 30-60% of the total grain
an insoluble residue which contains structural nitrogen.
proteins, such as those of the cell wall. The prolamins from each species are given
In barley, wheat, maize, rye and sorghum, trivial names which are often used and which
the prolamin fraction makes up most of the are shown in Table 23.3. The amino acid com-
protein (Table 23.3). In rice, the most abundant position of the prolamins differs from species
types of proteins are glutelins, whilst in oats to species, but when compared to typical pro-
they are globulins. In cereal processing the teins they are all rich in glutamine and pro-
term gluten is often used to refer collectively line, and deficient in lysine and most other
to the endosperm proteins. charged amino acids. Tryptophan is absent
When the compositions of the different from zein but present in reasonable quantities
fractions are compared (Table 23.4) it is clear in hordein and gliadin. The glutelin fraction
that the low content of essential amino acids contains insoluble enzymes, ribosomal pro-
in most cereals (of which barley is typical) is teins and some membrane proteins.
Table 23.3 Percentage composition of the proteins from cereal seeds
Cereal NPN+ Prolamin Glutelin Insoluble Name of

albumin prolamin
+globulin
Barley 27.2 45.2 18.0 5.2 Hordein

Wheat 33.1 60.7 ---6.2--- Gliadin
Maize 6.8 55.4 22.9 Zein
Rye 26.4 40.5 24.1 4.3 Secalin
Sorghum 13.2 54.5 25.5 Kafirin
Rice 15.7 6.7 61.5 15.4
Oats 67 9 23 Avenin
Modified with permission from Bright, S.w.J. and Shewry, P.R. (1983)
Improvement of protein quality in cereals., CRC Critical Reviews in Plant
Science, 1, 49-93. Copyright CRC Press, Boca Raton, Florida. © 1983.
Seed composition 339
Table 23.4 Essential amino acid composition of • increasing the free amino acid content;
protein fractions from barley grain • modifying the structural genes coding for
Globulin Albumin Prolamin Glute/in
the storage proteins by site-specific mutage-
Amino acid (g amino acid kg protein-I) nesis to increase their lysine content.
Although the last approach is attractive, it is
ile 33 62 54 52
difficult to achieve. A number of factors con-
leu 68 86 69 87
lys 53 67 7 40 tribute to this:
met 15 24 13 19 • the proteins are encoded by complex multi-
phe 28 51 30 36
gene families;
thr 33 46 26 42
trp 8 15 8 13 • genetic manipulation of cereals (as with all
val 55 78 47 66 monocotyledons), especially gene transfer
arg 110 65 30 60 and regeneration, are very difficult to
his 18 25 13 25 achieve;
• modification must not prevent the normal
Modified with permission from Folkes, B.F. andYemm,
E.W. (1965) The amino acid content of the proteins of functions of the seed.
barley grains. Biochemical Journal, 62, 4--11.
Unusually, in rice the major storage protein is Legume proteins

also a glutelin. Legume proteins are of higher quality than
As the major storage proteins of oats and those of cereals, mainly because of their high-
rice (a globulin and a glutelin, respectively) are er lysine content (Table 23.2). The proteins,
relatively rich in lysine and threonine, their however, have a low content of the sulphur-
protein is of nutritionally higher quality than containing amino acids, cysteine and methion-
that of most other cereals. ine. Some legume seeds also contain toxic
The non-storage proteins, generally albu- compounds which are discussed in more
mins and globulins, constitute the basic struc- detail in Chapter 5.
tural and metabolic machinery of the cell. The Most proteins contain only 1-2% methion-
amino acid composition of these proteins is ine, but some plants contain sulphur-rich pro-
usually much better than that of the storage teins in their seeds. Brazil nuts and sunflower,
proteins. In both fractions, lysine and threo- for example, have proteins of molecular
nine make up over 3% of the proteins (Table weight 10 000-12 000 which contain 15-20%
23.4). The corresponding fractions from maize methionine and 8% cysteine. Introduction of
and wheat have compositions similar to those the Brazil nut genes into soyabean or other
of barley. legumes would be one way of increasing the
Many attempts have been made to improve sulphur content of the seed. Maize also con-
the nutritional quality of cereal proteins. The tains a small sulphur-rich protein, but it is pre-
strategies employed have included: sent in only very small amounts. It may also be
• reducing the proportions of nutritionally possible to increase expression of the gene for
poor storage proteins - some spontaneous this protein to increase the sulphur amino acid
high-lysine mutants of maize, such as content of maize.
opaque-2, have a reduced prolamin content
together with increased levels of lysine-rich
23.3.4 SEED MINERALS
proteins;
• increasing the proportion of lysine-rich Phytic acid (inositol hexaphosphate) is an
albumins and globulins - the high-lysine important component of cereal seeds (Figure
barley line, Hiproly, has increased levels of 23.1). It serves as a store of phosphate for the
metabolic proteins; seedling, but its phosphate groups carry nega-
tive charges which bind calcium, iron and zinc 23.4.1 STARCH BREAKDOWN
ions. This reduces the availability of these min-
In most seeds starch forms the main energy
erals to animals and can result in deficiency
reserve. It is degraded through the action of a
symptoms even when the diet appears ade-
variety of amylolytic enzymes to form simple
quate. The phosphate groups are removed
sugars which may be converted into sucrose.
from phytic acid through the action of an
A number of enzymes are potentially capa-
enzyme, phytase, which is produced by ger-
ble of contributing to starch breakdown.
minating seeds, fungi and microorganisms.
Thus, phytate in the diet of ruminants is bro- • a-Amylase hydrolyses a-1,4 bonds of amy-
ken down in the rumen and the phosphate is lose at random to produce a mixture of
made available. In non-ruminant animals, glucose and maltose. (Note that the names
however, up to two-thirds of the phosphate in a- and l3-amylase do not refer to the types
the diet may be in the form of phytate and be of glycosidic bonds hydrolysed.) a-Amy-
unavailable. This is not only nutritionally lase does not degrade maltose to glucose. It
important but also leads to excretion of phos- also attacks amylopectin but cannot
phate and pollution of the environment. hydrolyse a-1,6 bonds at branch points
and does not act well on bonds near to
branch points or near to the end of the
23.4 GERMINAnON
chain. It exists as groups of isoenzymes
When seeds germinate it is essential that the with different isoelectric points, some of
stored reserves are degraded to provide nutri- which may be able to attack native starch
ents at the correct time during embryo grains.
growth. This is achieved through hormonal • I3-Amylase is not a very widespread enzyme,
control mechanisms which increase the activ- being restricted to cereal seeds and a few
ity of proteolytic enzymes, starch-degrading other plants. It hydrolyses a-1,4 bonds of
enzymes, nucleases, phytase, etc. at the amylose at the penultimate position releas-
appropriate time. ing maltose. It also attacks amylopectin, but
Some of these enzymes are synthesized de stops two to three glucose units from branch
novo during germination but others are point and cannot hydrolyse a-1,6 bonds at
formed during seed development and are branch points. It eventually degrades amy-
stored, in an inert form, during seed matura- lopectin to limit dextrin, during which 5~0
tion. The latter type only require activation or % of the glucose is released. It cannot attack
release during germination. starch granules, and in many cases its pres-
ence does not appear to be essential for
starch breakdown to take place.
• a-Glucosidase hydrolyses a-1,4 bonds of
maltose and oligosaccharides.
• oligo-1,6-Glucosidase hydrolyses a-1,6
bonds in isomaltose and dextrins. It is pre-
sent in germinating seeds and, together
with a and l3-amylases, allows complete
degradation of starch by attacking the
branch points in dextrins released from
amylopectin.
• Starch phosphorylase is a widespread
enzyme in leaves and many storage organs.
Figure 23.1 Structure of phytic acid. Although present in some seeds it does not
Germination 341
appear to be of major importance in m~bi nate for a limited time, during which amy-
lizing their reserves. The product of actIon lases are produced and partial breakdown of
of the enzyme is glucose-I-phosphate. starch into simple sugars occurs. This process
is stopped by gentle heating of the grain
In most seeds, including cereals, a-amylase
(kilning) to produce malt. Peat may be added
and l3-amylase appear to be more important
to the fuel to impart characteristic flavours,
than phosphorylase, but starch breakdown in
for example in malt whisky production. The
pea seeds may be brought about by starch
rootlets of the germinated grain (culms) are
phosphorylase.
removed and may be used as cattle feed. Malt
Breakdown of starch reserves in wheat and
is ground and extracted by heating in water
barley has been extensively studied. It appe~rs
at about 65° C for several hours (mashing).
to be regulated by the plant hormone gIb-
This process allows starch to be degraded by
berellic acid (GA). This is produced by the
enzymes to form sugars and it gives rise to
embryo as it starts to grow, is released and
wort. If beer is being produced, hops are
passes to the aleurone layer, where it increas-
added and the wort is boiled to denature
es the activity of many hydrolytic enzymes.
enzymes, precipitate proteins and sterilize
These are then released into the starchy
the liquid. After cooling, brewer's yeast is
endosperm where the reserves are broken
added which ferments the sugars to produce
down. The presence of GA activates the genes
ethanol. The biochemistry of this process is
for a-amylase in the aleurone layer, leading to
described in Chapter 16.
increased synthesis and release. There are two
Malt whisky is produced using barley only
isoenzyme groups of a-amylase, one wit~ a
as the source of both enzymes and fer-
low isoelectric point (pI 4.5-5.0) and one wIth
mentable sugars. During this process no hops
a high pI (5.9-6.5). These differ in their capaci-
are added and the wort is not boiled. After fer-
ty to attack native starch grains a~d in the t~me
mentation the wash is fractionated by distilla-
at which they are produced dunng germma-
tion through several stills and the product is
tion. Similarly a-glucosidase and limit dextri-
stored in oak casks for at least 3 years, usually
nase also increase in activity in response to
longer. Grain whisky is made by adding
GA. In contrast, l3-amylase is present in the
cooked maize to malted barley as a source of
endosperm of the dry seed in an inactive form.
additional starch. The barley serves mainly as
During development of the seed it becom~s
a source of starch-degrading enzymes.
covalently linked to proteins in the protem
For making malt whisky, very stringent
bodies by formation of disulphide bridges. In
quality standards are set for the barley. The
this form it lacks enzyme activity, but the
grain acts as a source of both starch and amy-
active form is released during germination
lases which must be present in the correct pro-
when GA-induced proteolytic enzymes break
portions. In general, the starch and protein
down the proteins to which it is linked.
content of grain are inversely related to one
another. Thus grain with a high protein con-
23.4.2 BEER AND WHISKY PRODUCTION
tent will have a high amylase activity but a low
starch content and will be unacceptable for
About 20% of the barley grown in Britain is malt production. Maltsters normally accept
used for malting and hence for alcohol pro- only barley with a nitrogen content of less
duction. Alcohol production depends on the than about 1.7% nitrogen (on a dry matter
fermentation of simple sugars which are basis). The presence of large amounts of
released from stored starch when cereal nitrogenous constituents in malting barley
grains germinate. The initial stage is calle~ results in unacceptable flavours and may pro-
malting: barley grains are allowed to germI- duce a cloudiness or haze when used in beer
production, particularly when the beer is endopeptidases and carboxypeptidases with
chilled. High activities of l3-glucanase acidic pH optima (3-6).
enzymes, which attack glucans in cell walls, The production of proteases in cereals
are also desirable. Failure to break down the appears to be induced by GA. In addition to
walls slows down starch degradation during increasing protein breakdown, these enzymes
malting and leaves residues which may hinder may also release other enzymes, e.g. l3-amy-
filtration steps in brewing. lase, from their association with proteins.
In addition to its chemical composition, Hydrolysis of stored proteins releases a
maltsters also require grain with a high and mixture of amino acids. The balance of amino
uniform germination. This minimizes losses acids demanded by the developing tissues
resulting from some grains developing too far usually does not match that of the amino acids
and using their sugars as an energy source to released from stored protein, so a considerable
support their growth. Thus two-row barleys are amount of interconversion by transamination,
preferred because of their more uniform grain breakdown and resynthesis of carbon skele-
size. The speed and uniformity of germination tons takes place.
may be improved by addition of gibberellic acid
during malting.
23.4.4 LIPID BREAKDOWN
Hydrolysis of stored lipids produces fatty acids

23.4.3 PROTEIN BREAKDOWN
and glycerol. The glycerol is converted into
Plants contain many proteases which are dihydroxyacetone phosphate in the cytoplasm
required not only for germination, but also and then used mostly for synthesis of sucrose.
for protein turnover, which takes place in all Fatty acids are degraded by the l3-oxidation
plant tissues to a greater or lesser degree. In pathway (Chapter 13) to form acetyl-CoA
seeds, proteases are associated with the pro- which is used to synthesize carbohydrates
tein bodies, which function as specialized through use of the glyoxylate cycle (described
vacuoles. These enzymes are mainly acid in Chapter 11).
VEGETATIVE GROWTH OF PLANTS 24
24.2 Composition of shoots 344
24.2.1 Plant cell walls 344
24.3 Important shoot crops and their 347
products
24.3.1 Temperate grasses 347
24.3.2 Tropical grasses 348
24.3.3 Forage legumes 348
24.3.4 Brassicas 348
24.3.5 Straws 348
24.3.6 Hay 348
24.3.7 Sil~ge 349
24.3.8 Wlod 349
24.4 Important root and tuber crops 351
24.4.1 Root crops 351
24.4.2 Tubers 351
24.1 INTRODUCTION ents and for root-produced hormones such as

gibberellins and cytokinins, which in turn
Once the root system has developed and pho- stimulate shoot development.
tosynthesis is well established, seedlings are An important metabolic function of leaves
no longer dependent on nutrients supplied by is to carry out photosynthesis, providing a
the seed. However the functions of the root source of energy for the entire plant.
and the shoot are closely integrated and each However, leaves also control water loss, are
is dependent on the other for its survival. involved in nitrogen metabolism and storage,
The root system takes up water and miner- and undergo senescence. In addition to carry-
al elements from the soil. Some elements, ing out photosynthesis, chloroplasts also
such as nitrogen, may also be converted into assimilate ammonia produced in photorespi-
organic compounds in the root. The uptake ration, synthesize amino acids, and reduce sul-
and assimilation of materials from the soil phate and nitrite. Leaves serve as a temporary
requires energy and carbon skeletons which store for sugars produced in photosynthesis
are largely provided from the shoot in the (in the form of starch) and of protein (mainly
form of sucrose via the phloem. Some of in the form of Rubisco) which may later be
these sugars are oxidized in respiration, mobilized and contribute to the development
whilst others are used to make the carbon of storage organs.
skeletons themselves. Thus the root system The stem transports nutrients and water
relies on shoot-produced sugars. It may also and may carry out significant amounts of pho-
be dependent on shoot-produced hormones tosynthesis, and store nutrients. In some
to stimulate and regulate its growth. The plants, particularly at early stages of their
shoot, on the other hand, depends on the development, the shoot consists mainly of leaf
root system for a supply of water and nutri- tissues. In others, however, stem tissue may
344 Vegetative growth of plants
make up a considerable proportion of the released and these may induce the produc-
shoot, and some of this may become lignified tion of phytoalexins - a sort of primitive
and take on a structural role. This is reflected immune system.
in the high fibre content of such shoots. This • The composition of the cell wall has very
consists principally of the thickened walls of large effects on the ability of both rumi-
cells which provide support and allow trans- nants and non-ruminants to digest food-
port through the shoot to take place. stuffs. Changes in the wall occurring during
The components of the shoots of some development change the digestibility and
plants are of great commercial significance. palatability (fruit ripening, etc.) of both ani-
Leaves and stems are eaten by man in the mal and human foods.
form of green vegetables and salads, and by
At least 90% of the structural material of cell
animals in the form of herbage. They may be
walls of all higher plants is made up of the
used in the form of timber and fibre products
polysaccharides cellulose, hemicellulose and
for construction purposes, and as a source of
pectin, described in Chapter 3. The remaining
raw materials for fermentation and other
10% is made up of a glycoprotein called
industrial processes.
extensin. This protein is rich in the unusual
During vegetative growth some plants may
amino acid hydroxyproline and also in ala-
produce storage organs such as tubers or roots
nine, serine and threonine. It is very insoluble
which store carbohydrates and which are
and difficult to extract from the wall. Most of
important agricultural products.
the hydroxyproline residues are glycosylated
with arabinose-containing oligosaccharides.
24.2 COMPOSITION OF SHOOTS Lignins (Chapter 5) are also present in some
cell walls.
24.2.1 PLANT CELL WALLS
One of the characteristic features of plant
Linkage of cell wall components
cells is the presence of a cell wall surrounding
the plasma membrane. The properties of The cell wall of the sycamore maple (Acer
these walls are important for a number of rea- pseudoplatanus) has been very thoroughly
sons. studied and much is known about the detail of
its structure. The structure of the cell wall of
• Plants have no separate skeleton to provide other dicotyledons is thought to be similar.
support, and the cell wall provides struc-
The wall consists of cellulose microfibrils
tural rigidity through the development of
running through a matrix made up of the
turgor.
other polysaccharides (Figure 24.1). The
• The presence of a cell wall limits the growth
microfibrils tend to run parallel to one another
of the cell. Before plant cells can grow, the
and are cross-linked by the other sugars. The
cell wall must be weakened. One of the
following are important in maintaining this
important functions of auxins and possibly
three-dimensional structure:
also of other growth-stimulating plant hor-
mones is to initiate this weakening. • xyloglucan (hemicellulose) is hydrogen-
• Interaction with the cell wall is one of the bonded to cellulose fibrils via its glucan
first events in the attack of pathogens on backbone [1];
plant cells. The properties of the walls are • xyloglucan is linked through the reducing
therefore of major importance in determin- ends to the arabinogalactan (pectin) [2];
ing resistance or susceptibility of plants to • arabinogalactans are covalently linked to
disease. As pathogens attack the cell wall, rhamnogalacturonans (pectin) via rham-
fragments of the wall components are nose residues [3].
Composition of shoots 345
The rhamnogalacturonan therefore cross- by random polymerization of phenolic
links cellulose chains via arabinogalactans and monomers.
xyloglucans. The polymerization of p-coumaryl, conifer-
In grasses and possibly other monocotyle- yl and sinapyl alcohols to form lignin is initiat-
dons, single units of ferulic acid and p-coumar- ed through the action of cell-wall peroxidases
ic acids are esterified to the arabinose side which convert these compounds to aroxyl rad-
chains of the arabinoxylan fractions of the icals (Figure 24.2). The unpaired electron with-
hemicellulose. These feruloyl esters may be in these moves around the radical
involved in cross-link formation catalysed by (mesomerism). Covalent bond formation
peroxidase. between unpaired electrons in two radicals
It is thought that cell-wall protein may be leads to the formation of dimers. The nature of
cross-linked by the peroxidase-catalysed the bond formed depends on the exact posi-
oxidative coupling of tyrosyl residues to form tion of the unpaired electrons at the moment
isodityrosine. This may tighten the wall struc- of reaction. The products can themselves gen-
ture and limit cell expansion. erate further radicals so that complex poly-
mers are formed, containing structures such as
those shown in Figure 5.6. The formation of
Lignin
this network enmeshes the other wall compo-
The name lignin does not refer to a single nents, but may also result in formation of
chemical structure, but rather to a range of covalent bonds between lignin and polysac-
structures of high molecular weight formed charide molecules. Covalent bonding to the
Xyloglucan
Cellulose
+-+--- microfibril
Qt--'--+--- Cell wall protein
Arabinoga/actan
(pectin)
rhamnogalacturonan
(pectin)
Figure 24.1 Linkage of the components of a plant cell wall. The cellulose microfibrils are cross-linked by
xyloglucans. These are hydrogen bonded to the microfibrils [1] and covalently linked to arabinogalactans
[2]. The arabinogalactans are also covalently linked to rhamnogalacturonans [3].
arabinoxylans and glucuronides of the hemi- because of the high content of esters and the
cell uloses is common. low methoxyl group content of grasses. In
contrast, ether-type linkages to hemicellulose
are not restricted to the grasses and these are
Role of lignin in digestion of plant cell walls
more stable in alkaline conditions.
Lignin is the main factor limiting the
digestibility of forages. The bonds in lignin
Role of cell wall in control of cell expansion
are extremely resistant to enzymic and chem-
ical attack. The presence of lignin also Auxin treatment causes cells to expand. It acts
reduces the ability of microorganisms to by promoting pumping of protons from the
degrade cell-wall proteins and carbohy- cell into the cell wall, thus decreasing the wall
drates. In some types of forage, lignification pH. This acidification weakens bonds between
renders the material more indigestible than components of the hemicellulose fraction and,
in others. The lignins from grasses are very because the wall is subjected to turgor pres-
much more soluble in alkali than those from sure, allows the cellulose micro fibrils to move
wood or non-grass forages. This seems to be away from one another. This mechanism
Peroxidase
CH20H CH 20H CH 20H

I I I
.CH CH CH
I II II
CH CH CH
R,J)R' R,j;R' R,/Q~

OH O· OH
Figure 24.2 Polymerization of lignin free radicals. p-Coumaryl, coniferyl and sinapyl alcohols are convert-
ed to free radicals by the enzyme peroxidase. The unpaired electron moves rapidly around the free radi-
cal and may form bonds with other free radicals or polysaccharides.
Important shoot crops and their products 347
allows cells to expand perpendicular to the also become increasingly lignified towards
orientation of the micro fibrils but not parallel maturity. This decreases the digestibility of the
to them, so that the direction of cell expansion other nutrients, except for the soluble carbo-
can be regulated by the orientation of hydrates which are readily utilized.
microfibrils in the wall. Mature forages are of lower quality than
young ones. This results mainly from the
increase in the proportion of the forage con-
24.3 IMPORTANT SHOOT CROPS AND THEIR
sisting of stems and the rapid decrease in
PRODUCTS
digestibility of this fraction with age. Although
24.3.1 TEMPERATE GRASSES in many grasses the leaves assist the stems in
providing support and may become lignified
Grasses and other pasture plants are the main
with age, they usually lose their quality less
components of the diet of herbivores. The
quickly than stems. In most forage legumes
overall composition of grasses and green veg-
the stems support the plant and the leaves
etables are given in Figures 9.7 and 9.8, where
serve as metabolic organs, so the leaves do not
they are compared with the composition of
become highly lignified and do not lose quali-
cereal grains.
ty. In a few grasses, e.g. timothy grass (Phleum
pratense) and sugarcane, the stem may serve as
Carbohydrates
a storage organ so that it may have higher
In comparison with cereal grains, temperate quality than the leaves.
grasses have a much higher content of cellu-
lose, hemicellulose and pectin associated with
Proteins
cell walls and they usually contain little starch.
Instead the main storage carbohydrates are The amino acid composition of proteins from
the fructans, but there may also be appreciable different grasses varies very little. Up to 50% of
amounts of sucrose, fructose, glucose and the protein is made up of Rubisco, the enzyme
other small sugars. The chemical nature of responsible for fixation of CO 2 in photosynthe-
fructans is described in Chapter 3. They are sis. Grass proteins are of higher biological value
very soluble in water and tend to be found than seed proteins: they are rich in arginine
mainly in the stem. Their content in grasses and contain appreciable quantities of lysine.
may vary, being promoted by high light inten- The first limiting amino acid is methionine and
sity, high photosynthetic rates and low tem- the second is isoleucine. There is also some
perature. Under favourable conditions they non-protein nitrogen present, principally in the
may constitute to 30% of the dry matter. There form of free amino acids and their amides (e.g.
is marked diurnal variation in the levels. glutamine and asparagine). Nitrate may also be
The fructan content of grasses is very present under some circumstances and may be
important in determining both palatability toxic because of its reduction to nitrite in the
and suitability of the grass for silage making. rumen.
They serve as the main substrates for fermen-
tation occurring during ensiling. Both the fruc-
Lipids
tose liberated by hydrolysis of fructans, and
pentoses released by hydrolysis of hemicellu- The lipid content of grasses is usually low (less
lose, may be fermented to lactic acid during than 60 g kg-I dry matter). There is very little
ensiling (Chapter 16). triacylglycerol, most (60%) consists of glycol-
The cellulose and hemicellulose content of ipid which forms part of the membranes with-
grasses is variable, increasing as the plants in the leaf. Between 60 and 75% of the total
mature. In addition, the cell-wall components fatty acids are in the form of linolenic acid.
24.3.2 TROPICAL GRASSES of parts of the plant which may be eaten. Thus
cabbages are apical buds, Brussels sprouts are
In tropical grasses, starch is the main storage
axillary buds, cauliflower and broccoli are
carbohydrate and accumulates mainly in the
leaves. flowers, etc. In the human diet they are main-
ly used to add variety and as a source of fibre,
High temperature appears to be a major
vitamins (especially C and E) and minerals
factor which increases the rate of lignification.
(e.g. calcium). The presence of goitrogens and
In this context it is interesting that C4 grasses
are usually of lower nutritional value than C3 S-methylcysteine sulphoxide (Chapters 3 and
5) may affect animals eating large quantities of
ones (tropical forages in general are of lower
brassicas.
nutritional value). Tropical grasses have a low
protein content which appears to be an intrin-
sic part of C4 metabolism. 24.3.5 STRAWS
Sugarcane is an example of a tropical grass
Straws consist of the stems and leaves of cere-
which stores sucrose in the stem. It provides
als or legumes which remain after collection
about 65% of the world sugar supply and, as a
of the seed. They all have high fibre and
result of breeding programmes, is very high
lignin contents and are of low nutritive value.
yielding. It is the most efficient of crop plants
Their use is restricted to ruminants. The
in converting solar energy into food.
dietary intake of straws is low unless they are
improved by addition of nitrogen in the form
24.3.3 FORAGE LEGUMES of protein or urea. Legume straws have a
Legumes also form valuable forage crops. In higher protein and mineral content than
pastures, clovers may be common. Clovers are cereal straws.
nutritionally superior to grasses in their pro- The digestibility of straws may be
tein content, and in some minerals (calcium, improved by treatment with alkalis. This
phosphorus, magnesium, cobalt and copper), breaks ester bonds between lignin and cell-
and their nutritive value falls little with age. wall polysaccharides, making the carbohy-
Their inclusion in the diet also increases drates more available. Sodium hydroxide or
palatability and feed intake. Sucrose and ammonia are usually used, and in the latter
starch are the main carbohydrates; fructans case the added nitrogen also increases the
are virtually absent. crude protein content.
A number of nutritional disorders may result
from ingestion of legumes. Bloat, resulting from 24.3.6 HAY
sheep and cattle grazing on clover- or luceme-
dominated pastures, is caused by inadequate Hay-making provides a means of storing
eructation of fermentation gasses as a result of herbage by reducing its moisture content.
foam formation caused by soluble legume leaf Fresh herbage typically has a moisture content
proteins. Oestrogenic compounds in some of 650-850 g kg-I but this must be reduced to
legumes lead to infertility in sheep. Some may less than about 150 g kg-I if the product is to be
contain toxic amino acids or vitamin K antago- stored successfully. This low moisture content
nists which cause sweet clover disease. inhibits respiration and microbial growth and
so minimizes loss of nutrients from the hay
during storage.
24.3.4 BRASSICAS
Some loss of nutrients may take place dur-
Different varieties of brassicas are grown for ing drying. Thus, plant enzymes may degrade
human consumption or as forage crops. The fructans to fructose and some sugars may be
versatility of some species is seen in the variety respired before the moisture content has been
Important shoot crops and their products 349
reduced sufficiently. In addition, microbial The exact course of the fermentation
spoilage, oxidation and leaching of soluble depends on the nature of the crop, its moisture
materials may also occur if the drying process content, exclusion of air, etc. Well-made silage
is prolonged. The protein and mineral content has a pH around 4 and a high lactic acid con-
of legume hays are higher than those of cereal tent. Good silage also usually contains acetic
hays. Overheating during storage of hays may acid, smaller amounts of propionic and butyric
lead to loss of protein as a result of cross-link- acids and some ethanol. Most of the protein of
ing of amino acids, especially lysine, to cell- the original crop is hydrolysed to amino acids
wall carbohydrates. Treatment of hays with but these are not extensively deaminated, so
preservatives, such as propionic acid, reduces the ammonia content is low. If anaerobic con-
fungal growth and allows them to be stored at ditions are maintained little loss of energy or
moisture contents of up to 500 g kg-I. dry matter takes place during ensiling. Badly
preserved silage, in which fermentation by
Clostridia or Enterobacter predominates, often
24.3.7 SILAGE results when the moisture content is too high.
Silage is produced by controlled fermentation It is usually only weakly acidic (pH 5-7) and
of a green crop of high moisture content. The the predominant acids are acetic or butyric.
aim of ensiling is to encourage the growth of Substantial degradation of amino acids to form
microorganisms which bring about fermenta- ammonia or toxic amines may occur.
tion of sugars, converting them to organic acids. The quality of the fermentation process may
This lowers the pH and so allows storage with- be improved by the use of inoculants of lactic
out spoiling. During the fermentation process acid bacteria. Selective inhibition of fermenta-
anaerobic conditions must be maintained and tion by other types of bacteria can be achieved
the pH must be reduced sufficiently to mini- through addition of acids such as formic acid,
mize growth of undesirable microorganisms. possibly together with formaldehyde.
The fermentation pathways which function
during ensiling are described in Chapter 16. 24.3.8 WOOD
The principal bacteria active in conditions Wood is made up of cells with heavily thick-
in the silo are lactic acid bacteria. These are fac- ened and lignified walls and with very little
ultative anaerobes which can survive under cell content. In softwoods the cells are mainly
aerobic or anaerobic conditions. Under anaer- tracheids, which provide both conduction and
obic conditions they ferment water-soluble support. Hardwoods contain a mixture of ves-
carbohydrates to organic acids, mainly lactic sels, which carry out conduction, and fibres,
acid. Some degradation of hemicellulose which provide support. Because hardwoods
occurs, yielding pentose sugars which may have fewer fibre-like cells and these tend to be
also be fermented to lactic or acetic acid. They shorter than in softwoods, they yield weaker
grow rapidly under acidic conditions. Strictly papers. They may be used for high quality
anaerobic Clostridia may also be present, and writing papers and have attractive decorative
some species ferment sugars to butyric acid, or properties. Wood at the centre of the stem
ferment amino acids to acetic and butyric (heartwood) is often darker and harder than
acids, ammonia and amines. Enterobacteria that at the outside (sapwood). The cells in the
ferment soluble sugars to ethanol, acetic acid heartwood are dead and contain extractives,
and hydrogen. They are facultative anaerobes some of which are polyphenolics, and which
and therefore compete with lactic acid bacte- penetrate the walls and lumen of the cells.
ria. However their growth is optimal at pH 7 These give the heartwood its darker colour and
and their activity is significant only at the start make it aromatic as well as making the cells
of the fermentation process. more resistant to attack by fungi and insects.
Wood consists primarily of cell walls and to those extracted by organic solvents. Many
thus contains cellulose, hemicellulose and different compounds may be present. Some
lignin. Typical values for the gross composi- resins, such as frankincense, are produced
tion of softwoods and hardwoods are given in commercially.
Table 24.1. The differences are relatively small, In different species of softwoods, resins may
although hardwoods tend to contain slightly contain resin acids (which are present in tur-
more cellulose and less lignin. The structure of pentine), fatty acids, sterols and polyphenolics.
hemicellulose molecules varies widely Hardwood extractives include mono- and
between species. However, in general, the triterpenoids, triacylglycerols and polypheno-
main constituent of softwood hemicellulose is lics. These compounds may be responsible for
mannose, followed by xylose, glucose, galac- the durability and colour of the wood, and may
tose and arabinose. In hardwoods xylose is by be of value in the tanning industry.
far the most abundant monomer, followed by
mannose, glucose and galactose with small
Paper making
amounts of arabinose and rhamnose.
As noted in Chapter 5, lignin of softwood This depends on the reduction of wood to its
trees is formed by polymerization of coniferyl constituent fibres (pulping). Paper is made
alcohol units whilst that of hardwoods is from pulp by depositing the suspension of
formed from both coniferyl and sinapyl alco- fibres on a moving screen to form a web, in
hol groups (see Table 5.1). Hardwood lignin which the fibres are randomly aligned and
thus has a higher methoxy (-OCH 3) group interwoven. The web is then pressed and
content (18-22%) than softwood lignin dried to produce paper, in which the fibres are
(12-16%). Hardwood lignins are also of lower held together by hydrogen bonds.
molecular weight, possibly because syringyl Pulp can be prepared by mechanical or
groups cannot cross link at the 5 position chemical methods. Mechanical pulps are made
because of the extra methoxy group which by compression and grinding of wood chips,
they contain. Carbon-carbon (-C-C-) bonds combined with heat treatment. They are chem-
are much more difficult to break than ether ically unaltered and thus have a high lignin
(-C-O-) bonds. The lower proportion of content. They tend to be bulky and porous and
-C-C- bonds in hardwood lignin and its lower cannot be bleached to yield a very white prod-
molecular weight makes it pulp more rapidly uct. They also tend to yellow when exposed to
than softwood lignin. light. This process produces paper which is
well suited for printing of newspapers, etc.
Chemical pulping aims selectively to
Wood extractives
remove lignin but there is also some loss of
These can be extracted with non-polar and carbohydrates, especially hemicellulose, so
polar solvents. The term resins is used to refer that yields of pulp are lower than those
obtained by mechanical pulping. Chemical
Table 24.1 Gross composition of wood from hard- pulping may be achieved by sulphite pulping
wood and softwood trees
or, more commonly, by the Kraft process. In
Substance Softwoods (%) Hardwoods (%) sulphite pulping, chips are heated with solu-
tions of sulphur dioxide at various pH values.
Cellulose 42 45 This cleaves ether bonds in the lignin and
Hemicellulose 27 30 sulphonates the products to give rise to small,
Lignin 28 20 soluble compounds. Some of these ligno-
Extractives 3 5 sulphonates are used as pellet binders in ani-
Reproduced with permission from: Walker, J.c.P. (1993) mal feedstuffs. In Kraft processing, solutions
Primary Wood Processing, Chapman & Hall, London. of sodium hydroxide and sodium sulphide are
Important root and tuber crops 351
used. These also cleave ether bonds and portant but in others (e.g. radish) most of the
degrade the lignin, but they are less selective growth occurs in the hypocotyl and is above
than sulphite so there is greater loss of carbo- ground.
hydrates. Chemical pulps produced by these Root crops generally have a low protein
processes can be bleached to very high white- and dry matter content. A considerable part of
ness. Bleaching removes further lignin and the dry matter consists of sugars. In swedes
may be achieved by treatment with chlorine, and turnips the main sugar is glucose, where-
oxygen or chlorine dioxide. Use of chlorine as as in mangels, fodder beet and sugar beet it is
a bleaching agent has declined because of the sucrose. Sugar beet is grown principally for
need to minimize the environmental impact of sugar production and the residue after extrac-
organochlorine compounds produced. tion of the sucrose (sugar beet pulp) is used for
feeding to ruminants.
24.4 IMPORTANT ROOT AND TUBER CROPS
24.4.2 TUBERS
In addition to their roles in absorbing nutri-
ents and anchoring the plant to the ground, Important tubers include potatoes, cassava,
some roots have developed into storage sweet potatoes and yams. Tubers generally
organs. This is particularly the case with bien- have a higher dry matter and a lower fibre
nials and herbaceous plants, allowing them to content than root crops. They also differ in
overwinter successfully. Grass roots are also having starch as their main storage reserve
important in storing reserves to supply the instead of free sugars.
needs of the growing shoot in spring. The
stored reserves in the roots of weeds such as
Potato
bracken, docks and dandelions are central to
their survival and to the difficulty of eradicat- The potato is an important crop in Europe,
ing them. Tubers, such as those of the potato, where 75% of world production takes place.
are underground storage organs which are Cool nights and warm days promote develop-
formed by swelling of specialized under- ment of its tubers. Typically 70% of the dry
ground stems called stolons and they there- matter of the tuber consists of starch. Some
fore exhibit many of the characteristics of phosphorus is associated with potato starch,
stems. Roots and tubers are important crops which makes it more viscous than most starch-
and are used as both human and animal foods. es and gives it specific industrial uses. About
6% of the dry matter is protein, and there are
substantial quantities of low-molecular-weight
24.4.1 ROOT CROPS
nitrogen compounds, including free amino
True roots make a small contribution to the acids. The quality of the protein is relatively
human diet but are widely used in animal high, being quite rich in lysine but it is limited
feeds. Thus carrots, parsnips, radish and beet- by its methionine and cysteine content.
root are used as human food, and various root Potatoes contain several alkaloids, of which
crops including turnips and swedes (Brassica the most important are derivatives of solani-
spp.) and mangels and fodder beet (forms of dine (see Chapter 5). These compounds are
Beta vulgaris) provide animal feed. Sugar beet toxic when they occur at levels in excess of
provides 35% of the world sugar supply, and about 0.15 mg g-l and environmental factors
also produces by-products which are used as such as exposure to light and sprouting may
animal feeds. The storage organ arises partly promote their accumulation.
from swelling of the tap root and partly from Because of their low fibre content potatoes
the hypocotyl. In some instances (e.g. carrots are suitable for feeding to pigs and poultry,
and parsnips) the hypocotyl is relatively unim- however the protein in uncooked tubers has a
very low digestibility because of the presence of Maillard reaction and may result in develop-
a protease inhibitor which prevents digestion. ment of an unacceptable degree of browning.
As the inhibitor is denatured by heat, it is nor- Potatoes to be used for making crisps must
mal to cook potatoes before feeding to these contain no more than 2.5-3 mg reducing
animals. This is not necessary for ruminants sugar g.! fresh weight. Unfavourable storage
because the inhibitor is destroyed in the rumen. conditions, especially storage of tubers at
temperatures below about 10° C, may sub-
stantially increase the concentrations of both
Browning reactions
sucrose and reducing sugars. This process,
Plant products are subject to several types of referred to as low-temperature sweetening,
'browning reactions'. These occur in potatoes results from changes in the relative propor-
and are also common in other plants. Brown tions of starch and free sugars because of dif-
colours may arise in plant products in several ferences in sensitivity to temperature of some
ways. In uncooked products the enzyme phe- of the enzymes involved in sugar metabo-
nolase may act on tyrosine to produce lism. A particularly important factor is the
quinones which undergo further, non- disproportionately great loss of activity of
enzymic oxidations, to form coloured products phosphofructokinase (PFK) as the tempera-
such as melanin. A range of non-enzymic ture is reduced, because of weakened interac-
browning reactions may also occur. In cooked tion between the subunits of this allosteric
potatoes, for example, the phenolic com- enzyme. This inhibits glycolysis so that free
pound, chlorogenic acid, may react with iron sugars, produced by starch degradation,
to produce grey-coloured products. accumulate and are converted into sucrose.
Differences in the susceptibility of different
varieties are related to the amount of chloro-
Cassava (manioc)
genic acid which they contain.
The Maillard reaction may take place at Cassava is an important tropical perennial,
high temperatures between carbonyl groups tuber-bearing plant. It has a higher dry matter
of sugars and free amino groups of amino but lower protein content than potato tubers.
acids or proteins. The amino acid lysine is par- The tubers can be used to make tapioca. The
ticularly susceptible to such reactions. The tubers of some varieties contain cyanogenic
products are brown in colour and are mainly glucosides, including linamarin, which are
responsible for the coloration which accompa- broken down by hydroxy nitrile lyase, to form
nies frying, roasting and other cooking carried HCN (see Chapter 3). However, traditional
out at high temperature. In some instances the methods of preparation of foods from these
colour may be unacceptable and may also sig- tubers minimize the breakdown or wash the
nificantly reduce the availability of amino glucosides from the tubers.
acids in the food.
A considerable proportion of the potato
Sweet potato
crop is processed into crisps, chips and dried
products. The free sugar content of potato Like cassava, sweet potatoes have a higher dry
tubers is important in determining their suit- matter but lower protein content than potato
ability for high-temperature cooking. The tubers. They also have a high content of vita-
reducing sugars react with amino acids by the mins A, Band C.
REPRODUCTIVE GROWTH 25
25.1 Flowering 353
25.2 Fruit development and composition 353
25.3 Fruit ripening 354
25.3.1 Changes in colour 354
25.3.2 Changes in texture 354
25.3.3 Changes in flavour 355
25.3.4 Respiration in ripening fruit 355
25.4 Seed development 356
25.4.1 Starch biosynthesis 356
24.4.3 Biosynthesis of fats 357
25.1 FLOWERING Flower initiation may be thought of as a

switch which activates a pre-determined pro-
Most plants flower at some stage in their life. gramme of development leading automatical-
Flowers in the form of cut flowers or flowering ly to flower, seed and fruit production.
pot plants are of importance both in the horti- Dramatic changes in metabolism and redirec-
cultural industry and as sources of perfumes; tion of nutrients occur as flowers, seeds and
some, such as cauliflower and broccoli, may be their surrounding structures begin to develop.
eaten - cauliflower curd has a high protein
and carbohydrate content. However the major
25.2 FRUIT DEVELOPMENT AND
importance of flowers is in the fruit or seed
COMPOSITION
formation which follows.
In many plants, flowering takes place when Fruits develop from the tissues of the ovary.
a terminal vegetative bud, which normally They vary considerably in shape and in the
gives rise to leaves and stem, is converted into nature of the tissues which make up most of
a flower bud. The mechanisms which control the fruit - in many the pericarp predominates
this transition are complex and poorly under- and may develop in several layers, whilst in
stood. Environmental conditions may induce others the receptacle is the major structure.
flowering in many plants, e.g. exposure to par- The composition of a range of fruits is given in
ticular temperatures or day lengths, but the Table 25.l.
response to these stimuli is not uniform Most fruits have a relatively low dry matter,
between species. Flowering behaviour may protein and lipid content. Often between 60
also be influenced by nutrition; the application and 80% of the dry matter is made up of car-
of large amounts of nitrogen often favours bohydrate which consists of both polysaccha-
vegetative growth at the expense of flower ini- rides, such as starch, and simple sugars such as
tiation. It is believed that flower induction is glucose and sucrose. The relative proportions
mediated by plant hormones whose concen- of these may change during ripening and may
tration or distribution is influenced by envi- contribute to changes in flavour.
ronmental and other stimuli. This is discussed Many fruits also contain large quantities of
further in Chapter 27. fruit acids and some vitamins, especially vita-
354 Reproductive growth
Table 25.1 Composition of typical fruits in the ripe state
Fruit Water Carbohydrate Protein Fat Starch Sugars

(gkg (gkg (g kg fruit-I) (gkg (ripe)/starch (ripe)/sugars
fruit -1) fruit-I) fruit-I) (unripe) (%) (unripe) (%)
Orange * 861 85 11 1
Tomato 931 31 7 3
Banana * 751 232 12 3 6 2000
Apple* 845 118 4 1 5 99
*Based on flesh only

Water, carbohydrate, protein and fat content from The Composition of Foods, 5th edn. are reproduced with the permis-
sion of The Royal Society of Chemistry and the Controller of Her Majesty's Stationery Office.
min C. The nature of the fruit acids differs ars, whereas in bananas starch breakdown
from one type of fruit to another. Malate pre- results in a dramatic increase in free sugar lev-
dominates in apples, bananas, cherries, plums els during ripening (Table 25.1).
and pears, but citrate is the main acid in fruits
such as citrus, figs, raspberries and strawber-
25.3.1 CHANGES IN COLOUR
ries. Tomatoes and gooseberries contain
approximately equal amounts of malate and Ripening of fruits is often accompanied by a
citrate, and in grapes the principal acid is tar- change in colour from green to predominantly
trate (Figure 25.1). These acids are stored in the red or orange hues. This is due to degradation
cellular vacuoles in the flesh and contribute to of chlorophyll and increased synthesis and
the acidic taste of many fruits, particularly in accumulation of carotenoids (Chapter 8) in the
their unripe state. plastids, and of anthocyanins (Chapter 5) in
the vacuole. However this process does not
occur in all fruits - pears and kiwi fruit, for
25.3 FRUIT RIPENING
example, remain the same colour as they
As fruits ripen they undergo extensive changes ripen. The colour changes may be restricted to
in composition. Frequently there is a decrease the outer coat, as in apples, or occur through-
in starch and an increase in free sugars, a loss out the fruit, as in tomatoes.
of chlorophyll and an increase in yellow and
red pigments. A loss of cell-wall components,
25.3.2 CHANGES IN TEXTURE
particularly pectin, leads to softening of the
cells and changes in texture. In apples loss of Ripe fruits are usually much softer in texture
starch occurs without an increase in free sug- than unripe fruits. This is a result principally
COOH
COOH COOH
I I Hl-H
H-C-OH H-C-OH
I I Hol-cOOH
H-C-H HO-C-H I
H-C-H
booH booH
booH
Malic acid Tartaric acid
Citric acid
Figure 25.1 Structures of some acids which commonly occur in fruits.

Fruit ripening 355
of changes in the structure of the cell walls. fruit. In others there may be little or no carbo-
Often during ripening there is a decrease in hydrate reserve in the unripe state, and these
the polygalacturonic acid content of the mid- must rely on sugars which are imported into
dle lamella of the cell walls. This is the region the fruit during the ripening process itself.
which is shared by adjacent cells and which This is the case in melons and grapes which
cements them together. It consists mainly of cannot sweeten after detachment of the fruit
pectin made up of polygalacturonic acid from the plant. These different patterns of
residues. Some of the free CO 2 groups may be development have obvious implications for
linked to one another by formation of cross- fruit handling and storage.
links via Ca2 + ions, and others may be esteri- Whilst the content of complex sugars, fruit
fied by methyl groups. During ripening, the acids etc. generally decreases during ripening,
pectin may be degraded by polygalacturonase, that of proteins usually increases. This is in
thus weakening the wall. This enzyme acts on part because of the need to synthesize addi-
demethylated pectin. Demethylation of the tional enzymes which are required for the
pectin during ripening, through the action of ripening process itself. Thus the breakdown of
the enzyme pectin methylesterase, allows starch, chlorophyll and cell-wall components,
attack by polygalacturonase. The presence of and the synthesis of new pigments, all require
calcium in fruit delays softening and prolongs the production of additional enzymes.
storage life, probably by preventing degrada- In many fruits ripening is accompanied by
tion of pectin polymers, particularly in the the development of very characteristic aromas
middle lamella. Physiological disorders of and flavours. These are generally due to the
apple, such as bitter pit, appear to be associat- presence of specific combinations of volatile
ed with low calcium levels in the fruit and compounds such as esters. Their synthesis
occur because of poor transport of calcium into requires the activation of specific metabolic
the fruit. They can be alleviated by direct pathways.
application of calcium to the fruit, but not usu-
ally by application to the soil.
25.3.4 RESPIRATION IN RIPENING FRUIT
The flesh of many fruits contains stone cells
which have hard, highly lignified cell walls Biosynthetic activities in ripening fruit require
and which contribute to the texture of fruit energy, which is provided through respiration
such as pears. They also often contain tannins occurring in the fruit. Many fruits show very
which are bitter and which contribute the characteristic changes in respiration during
astringent flavour to fruits such as persimmon. ripening. In some, respiration shows a climac-
teric during which there is a rapid and short-
lived increase in the rate of respiration at the
25.3.3 CHANGES IN FLAVOUR
beginning of the ripening process (Figure
Generally, as fruits ripen there is a decrease in 25.2). In climacteric fruits the magnitude of the
acidity and an increase in sweetness. The increase in respiration rates varies widely. It is
decreased acidity often results from reduction about five-fold in avocado and three-fold in
in fruit acids, although in fruit such as bananas tomatoes and bananas, but no increase is
there is actually an increase in acid content detectable in citrus fruits, pineapples, grapes
during ripening. At the same time there is usu- etc. Ethylene appears to be produced and to
ally an increase in the concentration of simple influence ripening in climacteric fruits, but to
sugars such as sucrose and, less frequently, play no part in the normal ripening of non-cli-
glucose. In many cases these sugars are pro- macteric fruits. The ripening of many fruits
duced by metabolism of starch which is often can be slowed dramatically by removal of eth-
present in considerable quantities in unripe ylene from the storage environment. The role
356 Reproductive growth
of ethylene in control of ripening is discussed There is considerable interest in carrying
further in Chapter 27. out genetic manipulation to modify patterns
It is not clear why some fruits show a cli- of fruit ripening, and this is discussed in
macteric pattern of ripening whilst others do Chapter 27.
not. Climacteric and non-climacteric fruits do
not fall into groups of related plants, and no
25.4 SEED DEVELOPMENT
obvious biochemical characteristics have been
correlated with such patterns of development. Growing seeds are powerful sinks to which
Indeed in the case of tomato (which is normal- nutrients move from photosynthetic and other
lya climacteric fruit) there are non-climacteric tissues. As discussed in Chapter 23, the princi-
mutants which show no autocatalytic ethylene pal storage compounds in most seeds are
production, but which have otherwise identi- starch and proteins. In certain specific types of
cal compositions. seeds quantities of oils also accumulate.
Understanding of the processes occurring
during ripening has been helped by the exis-
25.4.1 STARCH BIOSYNTHESIS
tence of mutants. These lack specific enzymes
which are thought to be involved in the Starch consists of a mixture of the straight-
ripening process and they thus allow the chain molecule amylose, and the branched-
importance of the enzymes to be assessed. chain molecule amylopectin (see Chapter 3). It
Some, such as the nor and rin mutants of is found within plastids in the form of starch
tomato, lack the capacity to synthesize ethyl- grains or granules. Amylose normally makes
ene and are deficient in lycopene (red colour) up 15-30% and amylopectin 70-85% of the
and polygalacturonase. total, and in general the proportion of amylose
70
60
,
.s:::;
50
'0) 40
..>::
0'"
() 30 Banana
E
20
10
Apple
0
0 10 20 30 40 50
Days
Figure 25.2 Changes in respiration rates during ripening of climacteric fruits. The intensity and duration
of the increase in respiratory activity vary widely from species to species. In other fruit such as citrus,
pineapples and grapes no climacteric is seen. (Redrawn from Biale, J.B. (1950) Postharvest phYSiology
and biochemistry of fruits. Annual Review of Plant Physiology, 1, 183-206.)
Seed development 357
present increases with increasing maturity of leaves (Chapter 26). The pathways for biosyn-
the grain. thesis of the amino acids which are required to
Starch is synthesized from sucrose which make proteins have been described in Chapter
may initially accumulate in vacuoles. Some 20; they are very active in developing seeds.
invertase is present in starch-synthesizing tis- Seed storage proteins accumulate in protein
sues but its activity is rather low, and the bodies, which are membrane-surrounded
enzyme sucrose synthase is probably the main organelles, often spherical, with a diameter of
enzyme responsible for sucrose breakdown, as between 0.1 and 25 f.Lm.
the reaction which it catalyses is easily The genes coding for storage proteins are
reversible. The synthesis of starch takes place complex and the regulation of their expression
in the amyloplast and appears to be catalysed is imperfectly understood. Transcription of the
by starch synthase firmly bound to the starch nuclear genes gives rise to mRNA from which
granules. ADP-glucose acts as the glucose introns may be excised. The mRNA leaves the
donor. Glucose and fructose resulting from nucleus and is translated on the rough endo-
sucrose breakdown probably enter the amylo- plasmic reticulum, as the proteins produced
plast after conversion to triose phosphate. are targeted to the protein bodies. In the
Starch synthase catalyses the transfer of lumen of the endoplasmic reticulum the pro-
glucose residues from ADP-glucose to the teins are processed into their mature forms. In
non-reducing end of pre-existing primer mol- the case of legume storage proteins the modi-
ecules in the presence of K+ to produce a-l,4 fications are complex, involving hydrolysis of
bonds, but it is not known how the primers the polypeptide chain at specific points, the
themselves arise. It is possible that starch formation of disulphide bridges, and attach-
phosphorylase, which normally catalyses ment of sugar groups to some amino acid side
starch breakdown, may be used to synthesize chains (glycosylation). Some of these process-
short straight-chain oligomers of glucose. The es are ·brought about by the Golgi bodies.
action of starch synthase produces only linear, Processing of cereal protein pre-cursors is usu-
a-l,4-linked molecules. Branches are intro- ally simpler, not normally involving peptide
duced into such straight-chain molecules by hydrolysis or glycosylation. The prolamins are
branching enzyme. This hydrolyses a-l,4 deposited in different protein bodies from the
bonds and transfers the resulting short other storage proteins. In this case they may
oligosaccharides to a primary -OH group, thus pass directly from the endoplasmic reticulum
creating an a-l,6-linked branch. The action of to the protein bodies, whilst other proteins
branching enzyme therefore increases the pass through the Golgi bodies.
number of non-reducing chain ends where
starch synthase can act. Both starch synthase
25.4.3 BIOSYNTHESIS OF FATS
and branching enzyme exist in multiple forms,
but the significance of this is unknown. Oils are synthesized from sucrose transport-
ed in the phloem from photosynthetic tis-
sues. The glycerol and acetyl-CoA needed for
25.4.2 PROTEIN SYNTHESIS
lipid synthesis can be made from sucrose via
Seed storage proteins are synthesized from the glycolytic pathway. The conversion of
sucrose and a source of nitrogen such as gluta- acetyl-CoA into oleic acid takes place in the
mine or asparagine. Both of these are brought proplastids, as described in Chapter 19.
to the developing seed in the phloem. In many Modification of oleic acid and subsequent
plants, much of the nitrogen is formed by resynthesis of triacylglycerols occurs on the
mobilization of protein previously stored in the endoplasmic reticulum membranes.
PLANT NUTRITION 26
26.2 Biochemical functions of major plant 359
nutrients
26.2.1 Nitrogen 359
26.2.2 Sulphur 359
26.2.4 Potassium 362
26.2.5 Calcium 362
26.2.6 Magnesium 362
26.3 Trace elements - micronutrients 362
26.4 Toxic effects of minerals 362
26.5 Interaction between carbon and 363
nitrogen metabolism
26.5.1 Carbon assimilation 364
26.5.2 Nitrogen assimilation 364
26.5.3 Senescence and nutrient cycling 366
26.1 INTRODUCTION 26.2 BIOCHEMICAL FUNCTIONS OF MAJOR

PLANT NUTRIENTS
Plant growth depends on the presence of a
considerable number of elements. Some of 26.2.1 NITROGEN
these are required in large amounts, others in
Nitrogen is an extremely important element. It
only very small quantities. The presence of
forms a part of the structure of amino acids,
impurities in growth media may make it very
nucleic acids, alkaloids, etc. The pathways by
difficult to establish whether some elements
which inorganic nitrogen is incorporated into
are truly essential or not. It is usual to consid-
organic compounds are described in Chapter
er an element to be essential if a plant cannot
20, and the way in which it may be remobi-
complete its life cycle (i.e. produce viable
lized during filling of storage organs is dis-
seeds) without it, or if it has been shown to
cussed later in this chapter.
form part of a compound which is essential to
the plant. On this basis 17 essential elements,
26.2.2 SULPHUR
listed in Table 26.1, have been identified.
Those elements which are present in the Sulphur occurs mainly in the form of the
plant at concentrations of 1 g kg-lor more are amino acids cysteine and methionine, which
called the major elements or macronutrients, are constituents of many proteins. Cysteine is
whereas those present at 100 mg kg-I or less also found in glutathione, a tripeptide ('Y-glu-
are the trace elements or micronutrients. tamyl-cysteinyl-glycine), which serves as a
360 Plant nutrition
Table 26.1 Levels of essential minerals in and goitrin which cause goitre, and liver and
plant tissues kidney damage, in non-ruminants.
Element Concentration(mg kg-l
dry matter) Assimilation of sulphur
Hydrogen 60000 Sulphur is available to the plant mainly as
Carbon 450000 sulphate (SO~-). In plants and animals it is
Oxygen 450000 present mainly in its reduced (-SH) form so
Nitrogen 15000 that assimilation requires reduction. This
Potassium 10000 reduction cannot be carried out by animals,
Calcium 5000
which are therefore dependent on plants and
Magnesium 2000
Phosphorus 2000 microorganisms for a supply of reduced sul-
Sulphur 1000 phur in the form of cysteine and methionine.
Chlorine 100 Sulphate is absorbed from soil by the roots.
Iron 100 A small amount is reduced in the roots but
Boron 20 most is transported to the shoot, where it is
Manganese 50 reduced in the chloroplasts. The first step in
Zinc 20
the reduction involves the reaction of sulphate
Copper 6
Nickel with ATP to produce adenosine-5'-phospho-
Molybdenum 0.1 sulphate (APS) and pyrophosphate in a reac-
tion catalysed by ATP sulphurylase (Figure
Modified from: Stout, P.R. (1961) Proceedings of 26.1). The sulphate group of APS is then trans-
the Ninth Annual California Fertiliser Conference,
pp.21-23. ferred to the sulphur atom of another com-
pound (possibly glutathione) before it is
reduced using electrons supplied by reduced
ferredoxin. Sulphide results (possibly still
redox buffer, keeping -SH groups in their bound to glutathione) and this is rapidly con-
reduced state. verted into cysteine by reaction with 0-
Sulphur also forms part of several vitamins acetylserine (see Figure 26.1). Methionine and
and coenzymes e.g. CoA, lipoic acid, biotin other sulphur-containing compounds are
and thiamin (Chapter 8). Iron-sulphur pro- made from cysteine.
teins, such as ferredoxin and those involved
in electron transport, contain inorganic sul-
Sulphur deficiency
phur.
Of particular interest to agriculturalists is the Sulphur deficiency is quite unusual, as most
existence of volatile or toxic sulphur compounds soils contain adequate sulphate. It results in
in some plants. The characteristic flavours and inhibition of protein synthesis and accumula-
odours of onion, garlic and some brassicas is tion of amino acids which do not contain sul-
due to the mercaptans, sulphides and sulph- phur. Growth rates, especially of the shoot, are
oxides which they contain. Similar compounds reduced. Chlorosis occurs first in the young
contribute to the taint which develops in milk leaves because there is very little remobilization
exposed close to swedes and turnips. Toxic sul- of sulphur from old leaves. Young leaves thus
phur-containing compounds include glucos- depend on the roots for sulphur uptake.
inolates which are found in plants such as Sulphur deficiency could become more
oilseed rape. These may be converted into a common because of lowered S02 emissions
variety of toxic thiocyanates, isothiocyanates into the air and reduced use of sulphur-con-
Biochemical functions of major plant nutrients 361
(a)
ATP + 80/' ---+
o o +PPi
H H
Adenosine-5' -phosphosulphate
(AP8)
AP8 + X8H ---+ AMP + X-8-80 3'

(b)
(c)
(d) CH 3
--
6=0 SH
6 + 8 2'
6H2
I + acetate
6Hz H-C-NH z
Hl-NHz 600H
600H
Cysteine
O-acetyl serine
Figure 26.1 Reactions for the assimilation of sulphate into organic compounds. (a) APS is formed by reac-
tion of AIP with sulphate, catalysed by AIP sulphurylase; (b) the sulphate group of APS is transferred to
X, which may be glutathione; (c) the sulphate group is reduced to sulphide (5 2-); (d) sulphide reacts with
O-acetyl serine to form cysteine, from which other sulphur-containing compounds arise.
taining fertilizers, e.g. ammonium sulphate or Uptake into the plant occurs against a very
superphosphate. strong concentration gradient and therefore
depends on energy provided by respiration.
Phosphorus is taken up only in the form of the
26.2.3 PHOSPHORUS
ion H 2PO.j and hence rates of uptake are very
In soils, phosphorus is present almost exclu- pH-dependent.
sively as phosphate. Most is in the form of In plants, phosphorus exists as Pi or organic
inorganic orthophosphate (P) but some occurs phosphates such as glucose-6-phosphate,
as organic phosphate esters. Pi is released from ATP, phospholipids, DNA, RNA and phytic
these by the action of phosphatases, either acid. Normally Pi is much more abundant than
from plant roots or microorganisms. organic phosphorus. In plants which are defi-
362 Plant nutrition
cient in phosphorus, the reserve of Pi decreas- appears to play an important part in the
es but the organic phosphorus content response of cells of the root and stem to grav-
remains fairly constant. Conversion of Pi to ity (gravitropism). Calcium accumulates on
organic phosphates takes place very rapidly. the lower side of horizontal roots and on the
Phosphorus is metabolically very active and is upper side of stems, and in both cases inhibits
readily remobilized from older tissues. elongation of that side of the organ.
Phosphorus is needed for export of sugars Symptoms of deficiency of calcium are most
from the chloroplast. Phosphorus deficiency obvious in young, meristematic tissues.
results in reduced growth, reduced tillering in
cereals, and particularly in reduced fruit and
26.2.6 MAGNESIUM
seed development.
Magnesium is a component of chlorophyll
and deficiency leads to interveinal chlorosis,
26.2.4 POTASSIUM
which is first seen in older leaves. It is
Potassium is quantitatively the most impor- involved in transfer of phosphate groups as
tant cation in plants, and is the major ion the true substrate of many enzymes using
which maintains turgor. Changes in turgor ATP is a magnesium complex of ATP (Chapter
pressure resulting from movements of K+ are 8). In addition it activates other enzymes such
responsible for leaf movements and for the as RUBISCO and may playa role in control of
opening and closing of stomata in response to carbohydrate synthesis in chloroplasts.
changes in water availability.
Potassium is very mobile in the plant and is
26.3 TRACE ELEMENTS - MICRONUTRIENTS
readily redistributed from older to younger
tissues. It is the only essential mineral cation Trace elements or micronutrients are those
which is taken up by active transport. It acti- essential elements which are present in the
vates many enzymes e.g. starch synthetase, plant at concentrations of 100 mg kg-lor less.
and promotes the synthesis of enzymes such Their main functions are given in Table 26.2.
as RUBISCO, thus enhancing the rate of pho- In addition to the elements listed in the
tosynthesis. table, sodium and silicon appear to be
Deficiency of potassium causes chlorosis of required by certain species, and may prove to
leaves and weakens the stems of cereals. It also be needed by all. Other elements such as
leads to low cell pH and results in the produc- cobalt may be needed by symbiotic bacteria.
tion of toxic amines such as putrescine.
26.4 TOXIC EFFECTS OF MINERALS
26.2.5 CALCIUM
At concentrations above those adequate to
Most soils contain adequate calcium, which is meet the needs of plants, a number of miner-
absorbed as Ca2 +. Concentrations of calcium in als may be toxic. Many non-essential elements
the cytoplasm of cells are very low and most of are also toxic to plants. These include the met-
the calcium in the plant is present in vacuoles als lead, cadmium, silver, mercury and tin,
and cell walls. Some calcium may be bound to which often result from pollution, and alu-
calmodulin and may act as a second messen- minium, which is naturally abundant and
ger within cells. Calcium is essential for cell becomes available under acidic conditions.
elongation and division, and for maintenance Increased availability of aluminium is a major
of membrane permeability and of cell-wall factor which reduces plant growth in acidified
structure through cross-linking of pectin soils resulting from acid rain. Some plants
(Chapter 24). Redistribution of calcium have become resistant to toxic metals, appar-
Interaction between carbon and nitrogen metabolism 363
Table 26.2 Functions of trace elements in plants
Element Function
Fe Forms part of the structure of haem, cytochromes and non-haem iron proteins, also needed for
synthesis of chlorophyll
Mn Resembles Mg2+ and can replace it in some functions; component of indole acetic acid oxidase
and needed for photolysis of water by PSII; deficiency leads to interveinal necrosis seen in
young leaves
Cu Component of plastocyanin, cytochrome oxidase, ascorbic acid oxidase and polyphenol oxidase,
needed for the synthesis of tryptophan and therefore of indole acetic acid; also needed for
desaturation of fatty acids and for superoxide dismutase
Zn Component of carbonic anhydrase which catalyses conversion of bicarbonate into CO2, and of
many dehydrogenase enzymes e.g. glutamic dehydrogenase, lactic dehydrogenase, alcohol
dehydrogenase; needed for superoxide dismutase activity
Mo Component of nitrogenase and nitrate reductase, essential for nitrogen assimilation
B Needed for synthesis of uracil and production of UDP sugars and nucleic acids; cell division in
meristems is particularly affected by its deficiency; transport and synthesis of sucrose (which
requires UDPG) also reduced
Cl Needed for evolution of oxygen by PSII in photosynthesis; Cl' acts as a negatively charged
counterion to K+
Ni Needed for urea breakdown in tropical legumes
ently by production of chelating agents called bon compounds. These serve as a source of
phytochelatins, which are small peptides con- energy and carbon skeletons, to which nitro-
taining the sulphur amino acid cysteine. gen-containing groups may be attached. The
Binding of the metals to these sulphur atoms growth of storage organs depends on
renders the metal non-toxic. Other ions such resources supplied by both shoots and roots.
as selenium, although not essential and with- Nutritional demands of individual parts of the
out effects on the plants, may be accumulated plant, which vary from time to time, are met
by some plants which may then be toxic to ani- by a flow of nutrients from site to site. These
mals which eat them. flows may change in the short term, allowing
temporary stores of starch formed in leaves in
the light to be redistributed to other organs
26.5 INTERACTION BETWEEN CARBON AND
during darkness. Over longer time scales,
NITROGEN METABOLISM
stores of both nitrogen and carbon built up
Individual tissues or organs of the plant have during vegetative growth may later be redis-
specialized functions but they must work tributed to developing storage organs.
together throughout the life of the plant to To explain how these nutrient flows are
ensure its survival and efficient growth. controlled, the concept of 'sources' and 'sinks'
Carbon compounds derived from photosyn- within plants has been developed. Sources are
thesis taking place in the leaves and other those parts of the plant which can supply
green parts of the shoot are distributed to assimilates and sinks are those to which they
other plant organs. The root system, on the move. In most plants the leaves are the most
other hand, is responsible for the uptake and obvious source as a result of their photosyn-
distribution of sufficient soil-derived nutrients thetic capabilities, but storage organs support-
and water for the entire plant. The assimila- ing re-growth, and cotyledons and endosperm
tion of nitrogen and other nutrients in the supporting seedling growth, also act as
roots depends on the shoot for a supply of car- sources. Organs such as developing seeds,
364 Plant nutrition
tubers, roots, stems, flowers and young leaves, sists of nitrate as the roots have a very low
in which growth or storage are taking place, capacity to assimilate nitrogen. However most
may be regarded as sinks. Tissues may act as plants have some nitrate reductase activity in
sources or sinks over short or long periods of the roots and thus some organic nitrogen-con-
time and many may act in both capacities at taining compounds are found in the xylem. In
different times during growth. wheat and maize, up to approximately 80% of
the nitrate entering the plant passes unaltered
through the roots. In barley about 50% is con-
26.5.1 CARBON ASSIMILATION
verted to organic nitrogen in the roots, and in
Carbon assimilation takes place principally in most legumes even more is assimilated.
the leaves through photosynthesis. Rates of Usually one nitrogen-rich compound, which
photosynthesis in leaves depend on the leaf in most non-legumes is glutamine, predomi-
area, as this affects their ability to absorb light. nates in the xylem or phloem.
Newly emerged leaves have a small surface In legumes, although glutamine is the ini-
area but a rapid growth rate. They are there- tial product of nitrogen fixation, little is nor-
fore not able to produce all the assimilates mally found in the xylem of plants which are
required to support their own growth and actively fixing nitrogen. Instead it is normally
must import materials from larger leaves or converted mainly to the ureides, allantoin or
from seed storage reserves. This situation per- allantoic acid (in tropical legumes such as cow-
sists until the leaf reaches about one-third of pea or soyabean) or to asparagine (in temper-
its maximum size, when rates of assimilate ate legumes such as peas, clovers or lupins).
production approximate to the demands of When legumes assimilate nitrate, some may
the leaf. Leaves above one-third of maximum pass unaltered into the xylem and some may
size generally become net exporters of assimi- be reduced to asparagine or glutamine before
lates which may be used to support the entering the xylem. Thus in tropical legumes
growth of nearby, newly formed and expand- ureides only predominate in the xylem whilst
ing leaves, or may contribute to the filling of the plants are assimilating nitrogen principally
seeds or other storage organs. by nitrogen fixation. Both temperate and trop-
icallegumes transport a mixture of asparagine
and glutamine in the phloem.
26.5.2 NITROGEN ASSIMILATION
Nitrate reductase is unusual because it is
The biochemical processes which are responsi- one of the few well-characterized inducible
ble for the assimilation of inorganic nitrogen enzymes in higher plants. Its activity is low
into organic compounds are described in when nitrate is not available but increases
Chapter 20. Nitrogen comes from the soil in when nitrate is supplied. Roots have a finite
the form of nitrate or ammonia or, in the case capacity to reduce nitrate, and as more nitrate
of legumes, from nitrogen gas in the air. is applied increasing proportions pass unal-
Nitrate reductase and nitrite reductase may tered into the xylem.
be located mainly in the roots, mainly in the Wherever it occurs, the incorporation of
leaves, or divided between the two. The nature inorganic nitrogen into organic. compounds
of nitrogen-containing compounds found in requires a reducing agent (such as NADH,
the xylem of plants of different species thus NADPH or reduced ferredoxin) and a-ketoglu-
covers a very wide spectrum, reflecting these tarate. NADH, NADPH and a-ketoglutarate
metabolic differences (Figure 26.2). are produced by metabolism of sugars or by
Cocklebur (Xanthium pennsylvanicum) repre- diversion of photosynthetic products from the
sents one extreme of the spectrum. In this production of sugars. Thus the metabolism of
plant over 95% of nitrogen in the xylem con- nitrogen is a drain on the carbohydrate metab-
Interaction between carbon and nitrogen metabolism 365
o % of nitrogen in xylem sap

100
cocklebur Xanlhium vi
white clover
oat
barley
bean
white lupin
amino amides ureides

nitrate
acids
Figure 26.2 Nitrogen compounds in the xylem of a range of plants. In cocklebur almost all nitrate passes
through the roots unchanged. Cereals convert up to about half of the nitrate taken up to amino acids
and particularly to glutamine. In most legumes, in addition to reduction of nitrate, ureides may arise
from the products of nitrogen fixation. (Redrawn from Pate, J.5. (1973) Uptake, assimilation and transport
of nitrogen compounds by plants. Soil Biology and Biochemistry, 5,109-119.)
olism of the plant, and uptake of large amounts it reduces the amount of fermentable sugar
of nitrogen may deplete sugar reserves. available. Similarly, late application of nitro-
Where nitrogen is converted into organic gen to sugar beet depletes the sugar content
compounds in the roots, they must have a sup- which reduces quality.
ply of sugars available. This is achieved by the There is a constant flow and recycling of
transport of sucrose from the leaves to the nitrogen around the plant. Nitrogen taken up
roots in the phloem. In the roots sucrose is and assimilated by the roots passes in the
hydrolysed to glucose and fructose, which may xylem to the shoot, where it may be tem-
then be oxidized to provide NADH, NADPH porarily stored as protein. This may be
and a-ketoglutarate. Where most nitrogen degraded to provide amino acids which are
assimilation occurs in the leaves, less sucrose re-exported in the phloem to support growth
has to be transported to the root system. of other parts of the plant. In non-legumes
Assimilation of nitrogen through the action nitrogen is exported from the leaves mainly as
of nitrogenase also requires large amounts of glutamine, but in legumes there is often a
energy and carbon skeletons which must both mixture of glutamine and asparagine.
be provided by catabolism of sugars. Glutamine and asparagine may be used to
Because the metabolism of one depends on make amino acids by transamination and the
the other there is often a strong inverse corre- amino acids may be converted into protein.
lation between the carbohydrate and nitrogen Proteins undergo constant turnover and are
content of plants. For example, late application eventually broken down to re-form amino
of nitrogen fertilizers to barley increases the acids. These are transaminated to form keto
protein and decreases the starch content. Both acids which are subsequently oxidized. At the
of these are undesirable in barley intended for same time a-ketoglutarate is converted to glu-
malting - the former because of the produc- tamate (see Figure 14.1) which is available for
tion of nitrogen compounds which impart off- synthesis of new amino acids or for export to
flavours to the product, and the latter because other parts of the plant. The amino acid com-
366 Plant nutrition
position of leaves and the storage proteins in Rubisco and this enzyme is extensively
seeds, roots or bark differ considerably. Thus degraded as seeds fill. The photosynthetic
extensive amino acid interconversion accom- capacity of the leaves therefore declines. It has
panies the mobilization of nitrogen reserves. been estimated that up to 40% of seed nitro-
gen is derived from leaves and a further 20%
26.5.3 SENESCENCE AND NUTRIENT CYCLING from pods and endosperm. The remaining
nitrogen is assimilated during seed fill.
Protein turnover results in continuous synthe-
Such patterns of nitrogen flux are common-
sis of proteins from amino acids and their sub-
ly found in many monocarpic annual plants
sequent breakdown. Some proteins turn over
including cereals. The redistribution of nitro-
faster than others, in general enzymes turn
gen in cereals may be even greater than in
over faster than structural proteins. Half-lives
vary from a few hours to several days. In legumes, with up to 90% of the nitrogen found
growing leaves, proteins are synthesized in the mature plant being taken up from the
faster than they are broken down so that the soil by the time the plant is half-grown, and
net nitrogen content of the leaves increases, 85% of the nitrogen in wheat leaves being
but when the leaves become senescent there is transported to the developing grain. The pho-
a net loss of nitrogen as rates of breakdown tosynthetic tissues closest to the ear, including
exceed those of synthesis. This nitrogen may the glumes and flag leaf, normally provide
be transported and stored in other parts of the much of the nitrogen.
plant. Much of the nitrogen found in seeds, for In many perennial plants, nitrogen required
example, is taken up from the soil before flow- for seed development is obtained from leaves
ering, and may have been cycled several times without correlated senescence occurring. In
and into several different organs before reach- perennials, much of the nitrogen made avail-
ing the seeds. This point is illustrated by able when the leaves senesce in the autumn is
Figure 26.3 which shows the nitrogen content transported and stored in the crown and roots
of bean plants during their growth. (herbaceous perennials) or in the bark (woody
The nitrogen content of the leaves reaches a perennials). In the latter case arginine-rich pro-
maximum in the early stages of seed develop- teins are found in the bark. Stored protein is
ment and then declines as the seeds fill. Most used as a source of amino acids needed to sup-
of the nitrogen in leaves is in the form of port new growth in spring.
500 ... _...................................................................................................... ,
400
,
c
Q) 200
E
z
Leaves
100 Roots
o~ ____ ~ __ ~~ __-+____-+____ ~
1 June 1 July 1 Aug 1 Sept 1 Oct
Date
Figure 26.3 Nitrogen content of broad bean plants during growth. Nitrogen accumulates in the leaves
during vegetative growth; it is then mobilized and transported to the seeds where it is used to synthesize
storage proteins. (Modified from Emmerling, A. (1880) Ladw. Versuchsshtat, 24, 113)
REGULATION OF PLANT GROWTH AND 27
DEVELOPMENT

27.2 Responses to light 368
27.2.1 Effects on photosynthesis 368
27.2.2 Phytochrome-mediated responses 368
27.2.3 Other responses to light 370
27.3 Responses to temperature 370
27.3.1 Photosynthesis 370
27.3.2 Vernalization 370
27.4 Responses to atmosphere 370
27.5 Responses to stress 371
27.5.1 Temperature stress 371
27.5.2 Water stress 373
27.5.3 Salt stress 373
27.6 Nature of plant hormones 374
27.7 Auxins 374
27.7.1 Biochemistry of auxins 374
27.7.2 Synthetic auxins 375
27.7.3 Sites of synthesis and transport of 376
auxins
27.7.4 Physiological activities and 377
applications of auxins
27.8 Gibberellins 379
27.8.1 Biochemistry of gibberellins 379
gibberellins
applications of gibberellins
27.8.4 Growth retardants 382
27.9 Cytokinins 384
27.9.1 Biochemistry of cytokinins 384
cytokinins
applications of cytokinins
27.10 Abscisic acid (ABA) 387
27.10.1 Biochemistry of abscisic acid 387
abscisic acid
applications of abscisic acid
27.11 Ethylene 388
27.11.1 Biochemistry of ethylene 388
368 Regulation of plant growth and development
ethylene
27.11.3 Methods of modulating the effects of 389
ethylene on plants
applications of ethylene
27.12 Miscellaneous plant growth regulators 392
27.12.1 Morphactins 392
27.12.2 Maleic hydrazide 392
27.12.3 Glyphosine 392
27.1 INTRODUCTION light. In C4 plants, rates of photosynthesis

increase almost linearly right up to full sunlight
Plants must regulate and integrate the activi-
intensity. For many plants growing at high
ties of all their parts. They must also respond to
light intensities, the amount of Rubisco appears
changes in their environment and do so in a
bewildering variety of ways. To achieve this to be rate-limiting and is only just sufficient to
account for observed rates of carbon fixation.
they have complex mechanisms for perception
C4 plants have the highest photosynthetic
of external events and for controlling their
rates and CAM plants the lowest. Alpine and
growth and development. Some aspects of
arctic plants must grow rapidly in the short
these control mechanisms, including the role of
growing seasons, and have rates of photosyn-
plant hormones, are considered in this chapter.
thesis which greatly exceed those of respiration.
Leaves of plants which grow in shade direct
27.2 RESPONSES TO LIGHT more of their biochemical activity to trapping
light and less to carbon dioxide fixation. They
The intensity, duration and quality of light to
have high chlorophyll contents, large, irregu-
which plants are exposed varies greatly at dif-
larly orientated grana stacks, and low levels of
ferent locations, depending on latitude in a
Rubisco and other photosynthetic enzymes.
predictable manner, and less predictably on
For crops in the field, the light intensity at
factors such as weather. The responses of
the lower leaves may be much lower than at
plants to light are extremely complex but can
the upper leaves, so most crops are never sat-
be classified into the groups described below
urated by sunlight when grown at normal
on the basis of the action spectra and the
plant densities.
intensities of light required.
27.2.1 EFFECTS ON PHOTOSYNTHESIS 27.2.2 PHYTOCHROME-MEDIATED RESPONSES
The action spectrum for photosynthesis is simi- Plant responses which can be brought about
lar to the absorption spectra of the chlorophylls, by red light but reversed by far-red light, or
with peaks in the blue and red regions. For vice versa, often involve phytochrome. Such
most C3 plants at normal carbon dioxide con- responses include photoperiodism, control of
centrations, as light intensity is increased pho- stem extension, apical dominance, seed germi-
tosynthetic rates increase until a limiting value nation and synthesis of phenolics.
is reached. This is called light saturation, and Phytochrome is a pigment-protein com-
for many C3 crops is reached at an intensity plex which exists in two forms. One of these
between a half and a quarter of normal sun- (Pr) absorbs red light whilst the other (Pfr)
Responses to light 369
absorbs mainly far-red light. Absorption of light with R:FR ratio of between 3 and 6, and
red light by Pr converts it to Pfr, whilst incandescent bulbs a ratio of about 0.7-0.8. At
absorption of far-red light by Pfr converts it to dawn or dusk, when the sun is less than 10°
Pro Pr is blue and shows an absorption maxi- above the horizon, the light is enriched in far-
mum at about 666 nm and Pfr is olive green red light as a result of passage of light through
and absorbs at about 730 nm. Spectra of the a longer air path which preferentially scatters
two forms are shown in Figure 27.1a. As both shorter wavelengths. Leaves absorb mainly
Pr and Pfr absorb red light, this light estab- blue and red light and allow green and far-red
lishes a steady state in which about 75-80% of to pass. Thus light in the shade of leaves is
the total phytochrome exists as Pfr. Far-red considerably enriched in far-red light com-
light, on the other hand, is absorbed only by pared with sunlight. In the shade of opaque
Pfr and therefore causes almost complete con- objects, most light comes from the blue sky
version of phytochrome to Pr. and so it is slightly enriched in blue.
Light from most sources contains both red Phytochrome is a dimer of two identical
and far-red light and establishes an equilibri- polypeptide chains each of about 120 kDa.
um between the different forms of phy- Each chain carries a chromophore group
tochrome. In normal daylight the ratio of red attached via a cysteine S atom. When light is
to far-red light (R:FR) is about 1.15. In compar- absorbed the chromophore changes from the
ison, white fluorescent light tubes give out cis to the trans form, or vice versa, and this in
(a)
0.45
0.4
0.35 Pr
0.3
.l'l
~
of!
0.25
j 0.2
0.15
0.1
0.05
200 300 400 500 600 700 800
Wavelength (nm)
(b)
red light
Synthesis - Pr ~ Pfr - Responses
~~ dark reversion Destruction
Figure 27.1 (a) The absorption spectra of the red- and far-red-absorbing forms of phytochrome (Pr and
Pfr, respectively). Red light (A = approx. 660 nm) is absorbed by both forms, but far-red (A = approx. 730
nm) is absorbed only by the far-red form. (b) The synthesis, interconversion and destruction of phy-
tochrome as it occurs in most dicotyledons.
turn causes subtle changes in the conforma- same. Both need about 15 photons to fix one
tion of the protein. It appears that Pfr is the molecule of carbon dioxide. At lower tempera-
active form of phytochrome which triggers tures, C3 plants require only about 12 photons
positive or negative developmental responses. whilst C4 plants need 14 because extra ATP is
In most dicotyledons and gymnosperms, Pfr used in carbon dioxide fixation. Above about
may also be converted into Pr by the process of 30° C, C3 plants become less efficient because
'dark reversion'. This is a relatively slow, pH- of the operation of photorespiration.
dependent process which takes place over a
period of hours and which does not require
27.3.2 VERNALIZATION
light (Figure 27.1b). It occurs only very slowly
in monocotyledons. Winter cereals require exposure to low-tem-
perature (vernalization) to promote flowering.
Most biennials also require exposure to cold
27.2.3 OTHER RESPONSES TO LIGHT
(several days to several weeks at temperatures
Some responses of plants show action spectra just above freezing) without which they will
with peaks in the blue or long wavelength not flower. It is the bud which is the site of
ultraviolet (UV-A) regions of the spectrum. response. When buds from vernalized plants
Phototropism is one such response. The opti- are transferred to non-vernalized plants they
mum wavelength is often around 450 nm and flower. The vernalization effect can also be car-
is thought to result from absorption of light by ried over in cuttings, suggesting that the stim-
a flavoprotein. ulus is chemical in nature.
Some responses of plants require much
higher (but still less than the intensity of day-
27.4 RESPONSES TO ATMOSPHERE
light) light intensities than those in which
phytochrome is involved. These are known as Photosynthesis and respiration work in
'high irradiance responses' (HIR). They may opposition to one another and, although
involve light in the UV-A, red and far-red some respiration is required to provide ener-
regions of the spectrum. In many cases there gy which 'drives' the plant's metabolism,
may be cooperation between phytochrome beyond a certain level it reduces the produc-
and blue-absorbing pigments. Other respons- tivity of the crop.
es, such as the inhibition of mesocotyl elonga- The carbon dioxide content of the atmos-
tion in dark-grown oats, require even lower phere is about 0.035% or 350 IJ-mol mol-I. After
light intensities than phytochrome, the equiv- remaining stable at about 280 IJ-mol mol-I for
alent of 1 second of full moonlight may be many centuries, it has risen dramatically since
enough. Such responses are triggered by red about 1850 as a result of the burning of fossil
light but not reversed by far-red. fuels. Approximately 13% of the atmospheric
carbon dioxide is used in photosynthesis each
27.3 RESPONSES TO TEMPERATURE year, and about the same amount exchanges
with carbon dioxide dissolved in the oceans.
27.3.1 PHOTOSYNTHESIS
Carbon dioxide, and other greenhouse gases
Optimum temperatures for photosynthesis such as methane, absorb long-wavelength radi-
are often similar to the daytime temperature at ation. When radiation from the sun reaches the
which plants normally grow. C4 plants usually earth it is re-radiated at longer wavelength, and
have higher optimum temperatures than C3 accumulation of greenhouse gases in the
plants because of their lower rates of pho- atmosphere prevents loss of heat, thus increas-
torespiration. At temperatures of 10-25° C the ing the energy retained. Increased cloud cover
efficiencies of C3 and C4 plants are about the and dust reflect back solar radiation and cause
Responses to stress 371
a cooling effect. How these opposing factors injury). Others, typically from colder environ-
might affect specific locations or the entire plan- ments, may only be damaged when frost
et is of great interest to agriculturalists and is occurs (freezing injury). Plants may also be
the subject of much speculation. damaged when exposed to high temperatures.
Photosynthesis in C4 plants is usually satu-
rated by carbon dioxide levels close to or just
Freezing injury
above those in the atmosphere (350 f.1mol
mol-I). In contrast, raising the carbon dioxide During natural freezing, ice crystals form in
concentration of the atmosphere in which C3 the extracellular spaces and grow by addition
plants are growing decreases the rate of pho- of water from within the cells, which therefore
torespiration by competing with oxygen for become desiccated. This causes the cells to
Rubisco, leading to faster net photosynthetic contract and the concentration of solutes
rates. In greenhouses, especially in winter, sup- inside them to increase, reducing the freezing
plementation with carbon dioxide at concentra- point. In hardened plants there is usually no
tions of up to about 1000 f.1mol mol- I may be damage to cell membranes or organelles, and
used. Above this concentration stomata close when they thaw water is taken back into the
and photosynthesis is inhibited. tissues. In cold-susceptible plants the mem-
If carbon dioxide levels are decreased below branes are often damaged and become leaky
atmospheric levels, net rates of photosynthesis as the cells shrink, and they are not then able
decrease and become zero at the carbon diox- to take up water after thawing. When rapid
ide compensation point where the rates of freezing is induced experimentally the cell
photosynthesis and photorespiration are contents may freeze.
equal. For C4 plants this occurs when the car- Tolerance to extracellular ice formation may
bon dioxide concentration is between 0 and 5 depend on accumulation of non-damaging
f.1mol mol- I and for C3 plants between 35 and solutes within the cell (cryoprotectants or
45 f.1mol mol-I. (Figure 27.2). compatible solutes) which decrease the freez-
Oxygen is required for respiration. Normal ing point and limit water loss. Production of
dark respiration, carried out by most tissues, cold-stress proteins or changes in membrane
uses the mitochondrial electron transport chain structure also increase resistance.
and cytochrome oxidase as its last step. This One of the most crucial factors which may
enzyme has very high affinity for oxygen and is cause development of ice in plants is the pres-
saturated at concentrations well below the nor- ence of ice-nucleation sites. These may be
mal atmospheric level, so that changes in within the tissues or may be provided by bac-
atmospheric oxygen concentration have virtu- teria on the surface. As bacterial species differ
ally no effect on rates of respiration. However, in their capacity to act as nucleation sites, frost
rates of photorespiration are reduced by lower- damage may be reduced by spray application
ing oxygen concentrations as Rubisco has a low of cultures of bacteria with low nucleation
affinity for oxygen (Chapter 17). potential, which replace the naturally occur-
ring ones.
27.5 RESPONSES TO STRESS
27.5.1 TEMPERATURE STRESS Chilling injury
Low temperature is probably the most impor- Some plants may be damaged by exposure to
tant factor which limits plant distribution. lower than critical temperatures, even though
Many plants which normally grow in hot cli- it does not result in freezing. The symptoms of
mates may be injured below a critical temper- such injury seem to arise mostly from changes
ature of between about 0 and 20° C (chilling which take place in the cellular membranes at
35~------------------------------~
~
30
III
1: 25
<5
u
'0 20
E
..a.
c: 15
0
:;
)(
I;::: 10
~
u 5
maple
ti
c:
0
50 100 150 200 250
-5
CO2 concentration (/-lmol morl)
Figure 27.2 The effect of carbon dioxide concentration on net rates of photosynthesis. Photosynthesis in
C4 plants is almost saturated by atmospheric levels of CO 2 (350 J.Lmol mol-1) but rates of photosynthesis
carried out by C3 plants generally increase up to about 1000 J.Lmol mol- 1• At low CO 2 levels, net rates of
photosynthesis become zero for C4 plants between 0 and 5 J.Lmol mol-1 CO 2 and for C3 plants between 35
and 45 J.Lmol mol-1•
the critical temperature. Above the critical carbohydrate synthesis. High temperatures
temperature the membrane is in a fluid state, may result in decreased protein synthesis,
but below this temperature it becomes semi- increased cytoplasmic viscosity and loss of
crystalline. The critical temperature is influ- membrane semi-permeability. Membrane-
enced by the ratio of unsaturated to saturated dependent processes such as photosynthesis
fatty acids and by the sterol content. The are particularly sensitive, especially photosys-
changes affect the permeability of the mem- tern II. Photosynthetic ATP production is also
branes and the activity of enzymes embedded inhibited because of the release of free fatty
in them, and lead to redistribution of metabo- acids which act as uncouplers of the electron
lites and damage to membrane-dependent transport process.
processes such as photosynthesis and ATP Although the production of many proteins
production. Chilling-resistant or hardened is decreased at high temperatures, that of a
plants have increased levels of unsaturated specific set of proteins, the heat-shock proteins,
fatty acids which could account for their lower is increased in response to temperatures 10-15°
critical temperatures. C above the optimum growth temperatures.
The production of heat-shock proteins also
occurs in most other organisms. A 70-kDa pro-
High-temperature stress
tein seems to be common to most organisms,
Plants are adversely affected by high tempera- but plants may also produce smaller (15-27-
ture. Some effects may result from differences kDa) ones. Some heat-shock proteins are asso-
in the temperature sensitivity of individual ciated with particular organelles (the nucleus,
metabolic pathways. Thus in C3 plants, pho- chloroplast etc.) whilst others form cytoplasmic
torespiration becomes increasingly important aggregates. These proteins may protect pro-
in relation to photosynthesis as the tempera- teins and nucleic acids from damage, possibly
ture rises, reducing the plants' capacity for net by stabilizing their folding, and may also bind
Responses to stress 373
ions released as membranes change their per- responses of plants to stresses such as flood-
meability at high temperature. ing, mechanical stress and pollutants.
In barley and maize, synthesis of 60-70-kOa
proteins is induced by water stress and in
27.5.2 WATER STRESS
many plants osmotin (26 kOa) is produced in
Many plants are exposed to significant response to water stress as well as salt stress
degrees of water stress and suffer consider- (Section 27.5.3).
able water loss from their tissues. As the
water content falls the concentration of
27.5.3 SALT STRESS
solutes in tissues increases to the point where
damage to enzymes might occur. In some In hot, dry environments, salt often accumu-
plants, specific, non-damaging solutes accu- lates because of high rates of evaporation, and
mulate to high levels and this helps water many areas of irrigated land have become
retention. Such compounds include sucrose, saline for this reason. Soils close to the sea are
glycerol, mannitol, proline and betaine. also often highly saline. High salinity makes it
Proline levels may increase to between 10 and difficult for plants to obtain water from the soil
100 times their normal values. Other plants and to grow, because of the negative osmotic
simply lose large amounts (up to 70%) of potential of the soil. Salinity also requires the
their water but still survive. Even moderate plant to deal with toxic amounts of certain
drought may considerably reduce yields of ions, particularly sodium, chloride and car-
agricultural crops, especially if it coincides bonate. Some crops, e.g. beet, tomato and rye,
with particularly sensitive phases of the are salt tolerant but others, such as peas and
plants' development such as germination, onions, are not.
seedling growth or flowering. Rates of photo- Plants growing in saline soils may have dif-
synthesis may not recover for several days ficulty acquiring potassium, as sodium
after stress is relieved, and old leaves may be reduces its uptake. Salt-adapted plants show
shed and growth delayed. enhanced discrimination between potassium
Rates of photosynthesis, respiration, pro- and sodium, and the discrimination is
tein synthesis and nucleic acid synthesis are increased by calcium.
decreased by water stress: cell growth and cell Some salt-tolerant plants, e.g. mangroves
wall synthesis are particularly sensitive. Water and salt-tolerant wheat, exclude sodium and
stress reduces protein synthesis, acting at the chloride from their tissues. Others may take
level of translation, but it induces the synthe- up salt but exude it again. Many accumulate
sis of a small group of stress-specific proteins non-toxic compatible solutes such as proline,
and some hydrolytic enzymes such as a-amy- betaine, sugars, galactosyl glycerol, polyols or
lase and ribonuclease. A general effect of malate which help to maintain osmotic bal-
many types of stress, including water stress, is ance with the soil. Some adapted plants have
to increase concentrations of free amino acids. weakened cell walls, allowing faster growth
ABA levels increase, particularly in leaves, for the same osmotic potential, whilst in others
as water stress develops. This causes K+ to some enzymes are unusually stable at high salt
leave the guard cells and the stomata to close, concentrations.
reducing both rates of water loss and stem Salt-stressed plants contain reduced quan-
growth. Resistant varieties of some plants may tities of most proteins (because of reduced
have increased levels of ABA. Levels of ABA protein synthesis and increased proteolysis),
also increase in response to nutrient deficien- however, the production of certain specific
cies or excesses, chilling, waterlogging and proteins may be increased. Cultured tobacco
salinity. Ethylene may also play a role in cells synthesize new proteins in response to
increased salt levels. One of them, a 26-kOa monate and polyamines, when applied to
protein called osmotin, may make up more plants, have pronounced effects on their
than 10% of the total protein in stressed cells, growth. As it is not at present clear whether
and may facilitate solute accumulation or these have hormonal functions, they are not
make cells more resistant to osmotic stress. discussed further in this chapter.
Maize, rice and citrus also produce 2S-30-kOa A considerable number of synthetic com-
proteins in response to salt stress. pounds have been synthesized and shown to
modify the growth of plants. Such compounds
are commonly known as plant-growth regula-
27.6 NATURE OF PLANT HORMONES
tors (PGRs) and many are commercially
In differentiated organisms, including higher important in crop production. Many of them
plants, the activities of individual tissues and are synthetic analogues of hormones which
organs must be regulated so that they operate mimic their effects or interfere with their pro-
in a coherent manner. They must also be able duction or action.
to respond to environmental stimuli. Plant-
growth substances or plant hormones form
27.7 AUXINS
one element of such control processes.
Plant hormones are normally defined as Indole-3-acetic acid (IAA) is the main endoge-
naturally occurring compounds which influ- nous auxin. Some other endogenous com-
ence biochemical processes in plants at con- pounds also show auxin activity, e.g.
centrations far below those at which nutrients indoleacetonitrile and indole acetaldehyde,
or vitamins are active. In comparison with ani- but this is because they are converted into IAA
mal hormones, there are fewer plant hor- by the plant.
mones and each produces a very wide range Since the discovery of IAA a large number of
of effects and acts on many different tissues. synthetic organic compounds which also have
Some appear to act in tissues where they are auxin activity have been synthesized and used
synthesized as well as moving around the as herbicides and plant-growth regulators.
plant. There are also very pronounced pat-
terns of interaction between the different
27.7.1 BIOCHEMISTRY OF AUXINS
groups of hormones. To emphasize these dif-
ferences, plant hormones are sometimes IAA can be synthesized by plants from the
known as plant-growth substances. amino acid tryptophan. Although alternatives
Hormone concentrations might be influ- exist, in most plants the route via indole-3-
enced by rates of hormone synthesis, by trans- pyruvic acid is probably preferred (Figure 27.3).
port and uptake into target tissues, and by In tissues infected by Agrobacterium tumefaciens,
metabolism in these tissues to form inactive or the bacterium causing crown gall, tryptophan is
bound forms. The sensitivity of tissues to hor- converted to IAA via the intermediate indoleac-
mones may also vary as a result of changes in etamide (Section 27.9.3).
the numbers or types of receptors present in IAA can be oxidized to form methylene-
target tissues. Thus hormone action in plants oxindole (catalysed by the well characterized
is extremely complex and, on the whole, poor- peroxidase enzyme, IAA oxidase) or to oxin-
ly understood. dole-3-acetic acid. IAA may also be converted
The main groups of plant hormones are the into a number of conjugates, collectively
auxins, gibberellins, cytokinins, abscisic acid known as 'bound auxins', by conjugation with
and ethylene. In addition, a number of other inositol, glucose or amino acids such as aspar-
phytochemicals such as the brassinosteroids, tic acid (Figure 27.4). Bound auxins are inac-
dihydroconiferol, triacontanol, methyl jas- tive but may release IAA on hydrolysis and
Auxins 375
Tryptophan
W-CH,-C..,....H,
N
aCi~ ~
/~:-
Tryptamine
Indole-3-pyruvic
w
.. ~C02
decarboxylation
daamination
HrCHO
Indole-3-acetaldehyde
Indole-3-acetic acid
Figure 27.3 Alternative pathways for the biosynthesis of auxins in higher plants. The route via indole-3-
pyruvic acid is most common.
may be forms in which IAA is transported in When applied at higher concentrations, a

germinating seeds. wide range of auxins, including those shown
in Figure 27.5, act as herbicides. Symptoms of
auxin herbicide treatment are distorted
27.7.2 SYNTHETIC AUXINS
growth, epinastically curved leaves and split
A large number of synthetic compounds stems, many of which are probably a result of
(Figure 27.5) have been shown to have auxin stimulation of ethylene production.
activity. Compounds such as indole-butyric A major advantage of auxin herbicides is
acid (IBA), a-naphthalene acetic acid (NAA) that they are selective in their action. Cereals
and 2,4-dichlorophenoxyacetic acid (2,4-0) are and other monocotyledons are quite resistant
widely used as plant-growth regulators. These to them, but dicotyledons are usually very sen-
compounds are more often used than IAA as sitive. This selectivity depends mainly on dif-
they are cheaper and more persistent in the ferences in the nature and orientation of
plant because they are not attacked by IAA monocot and dicot leaves, so that the sprays
oxidase. may be retained and penetrate to different
Methylene oxindole
IAA oxidase
Oxindole-3-acetic acid
Indole-3-acetic acid
~HrCOrllIUCOSe
~N)
H
Indole acetyl glucose
C02H
I
A-----rcHrCO--NH~H
~ ...N ) CH2
I
H C02H
Indole acetyl aspartic acid
Figure 27.4 Auxin metabolites. Oxidation leads to irreversible loss of auxin activity, but conjugation
with sugars or amino acids produces bound auxins from which free auxin may be regenerated by
hydrolysis.
extents. In addition, differences in the metabo- 27.7.3 SITES OF SYNTHESIS AND TRANSPORT
lism of auxins between different species exist OF AUXINS
and contribute to their selectivity. The phe-
noxybutyric acids, e.g. MCPB and 2,4-0B, do Meristems, enlarging tissues including the
not themselves have auxin activity but in some shoot apex, enlarging leaves and opening
species they are degraded into the corre- buds, and developing fruits and seeds are
sponding phenoxyacetic acids, which are major sites of auxin production. Longitudinal
active, by the ~-oxidation pathway which movement of auxins is strongly polar and
removes pairs of carbon atoms from the fatty requires ATP. In both the root and the shoot,
acid side chains (Chapter 13). Most legumes, movement is generally downwards and quite
including peas and clover, are unable to bring slow, (about 1 cm h-1 ). Lateral movement of
about this conversion and are resistant, allow- auxin occurs in both roots and shoots and
ing weeds to be selectively killed in these gives rise to phototropic and gravitropic
crops. responses.
Auxins 377
WH2-CH~H~02H
O
O-CH~02H
CI CH 3
I
H
Indolebutyric acid
(2-methyl, 4-chlorophenoxy)acetic acid
(IBA)
O
O-CH~H2-CH~02H
CI CH 3
a Naphthaleneacetic acid 4-(2-methyl, 4-chlorophenoxy)butyric acid

NAA
~O-CH2-C02H O-CH~H~H~02H
O
CI~CI CI'- CI
(2,4-dichlorophenoxy)acetic acid
4-(2,4-dichlorophenoxy)butyric acid
2,4-0
2,4-0B
(2,4,5-trichlorophenoxy)acetic acid
2,4,5-T
Figure 27.5 Structures of a selection of synthetic auxins. IBA and NAA are commonly used as plant-
growth regulators whilst the phenoxy derivatives are mainly used as herbicides.
27.7.4 PHYSIOLOGICAL ACTIVITIES AND polysaccharides, or weakens hydrogen bonds

APPLICATIONS OF AUXINS between wall components and allows the wall
to be stretched.
Effects on stem elongation
Unilateral illumination of shoots causes
Auxins stimulate the elongation of stem sec- auxin to become asymmetrically distributed
tions, but not usually of intact stems which and results in curvature towards the light
receive sufficient auxin from the shoot apex to (phototropism). Light perception in the apex
promote optimal growth. results in reduced rates of transport from the
Binding of auxins to the plasma membrane apex on the illuminated side, so that the con-
of stem cells activates a proton pump, causing centration of auxin on the shaded side is
secretion of protons from the cell into the wall. increased relative to that on the illuminated
This activates enzymes which hydrolyse wall side, thus promoting its elongation.
Effects on cambial development grains which settle to the lower side of the sta-
tolith. This is believed to cause an asymmetri-
Both auxins and gibberellins stimulate cambial
cal distribution of Ca2 + ions which in turn
cell division, but their effect when applied
causes redistribution of IAA.
together is much greater than either applied
Callus may differentiate to form roots or
alone (synergism). In addition, auxin is essen-
shoots when exposed to auxins or cytokinins.
tial for further differentiation of cells to form
High auxin/cytokinin ratios promote root for-
xylem, whilst gibberellins are needed for
mation, whilst low ratios promote shoot for-
phloem formation.
mation.
Role in apical dominance
Effects on flowering
The presence of the shoot apex normally
inhibits growth of lateral buds (apical domi- Application of 2,4-D or NAA is used commer-
nance). In many plants removal of the shoot cially to induce and increase the uniformity of
apex allows the lateral buds to grow, but this flowering of pineapple. Auxins generally do
can be prevented by application of auxin to not promote, and may even inhibit, flowering
the cut stem. Thus auxins originating in the in most other species.
shoot apex are thought to inhibit the develop- Auxins favour female flower production in
ment of lateral buds. In contrast, root-pro- a number of plants, including cucumbers. The
duced cytokinins promote such development, effects of auxins on flowering may be due to
as application of cytokinins to some inhibited their induction of ethylene production, as
buds can stimulate their growth. ethephon or ethylene produce similar
responses (Section 27.11.4).
Effects on root development and
differentiation of callus Effects on fruit development
Auxins stimulate the production of lateral Auxins in pollen ensure that a rapid burst of
roots, thus promoting root branching. Both ovary growth, accompanied by abscission of
endogenous and exogenously applied auxins the stamens and petals, usually follows polli-
stimulate the development of adventitious nation (fruit set).
root primordia from the stem, causing rooting When flowers are not fertilized abscission
of cuttings. Synthetic auxins such as IBA of the flower usually occurs, but in some
(indole butyric acid), NAA (naphthalene acetic species this can be prevented by auxin appli-
acid) or naphthalene acetamide are widely cation. Many synthetic auxins increase fruit-
used in commercial rooting powders, IBA and set in fruit containing many ovules, e.g. fig,
NAA often being used together. Although strawberry, squash, tomato and aubergine.
auxins promote initiation of roots, they gener- This may be used to overcome pollination dif-
ally inhibit root elongation as the concentra- ficulties in indoor tomatoes, or depressed fruit
tion of auxin required for maximal root set due to low temperatures in the field.
growth is very low (approximately 10-10 M). Auxins may also cause abscission of flowers
Auxin accumulates on the lower side of of fruit trees. Several auxins, including NAA or
both horizontal roots and shoots, but whereas naphthalene acetamide, can be used to remove
it promotes elongation of that side of the some flowers and prevent development of
shoot, it probably inhibits that of the root. excessive numbers of fruit, which tend to be
Thus roots bend downwards whilst shoots small.
bend upwards. The gravitational stimulus is Stimulation of fruit development by auxin
detected by statoliths containing heavy starch in pollen lasts only a short time and continued
Gibberellins 379
development of the fruit depends on hor- 13-hydroxylation pathway, and GAl has been
mones, especially auxin, produced by the identified as the form which actually promotes
developing seed. shoot growth in these plants.
Mature apples and pears often fall off the GAs may be metabolized into a number of
tree just before harvesting because of low lev- derivatives such as glucosides and protein-GA
els of auxin in the fruit, but application of NAA conjugates, which may function as storage
or 2,4,5-trichlorophenoxypropionic acid may forms.
be used to prevent this. A number of plant-growth retardants inhib-
it specific steps in the pathway leading to the
biosynthesis of GA12-aldehyde (section 27.8.4).
27.8 GIBBERELLINS
The gibberellins (GAs) contain 19 or 20 carbon 27.8.2 SITES OF SYNTHESIS AND TRANSPORT
atoms and are based on the ent-gibberellane OF GIBBERELLINS
ring structure (Figure 27.6). They have been
given numbers such as gibberellin AI (GAl) GAs appear to be synthesized in leaf primor-
etc., approximately in order of their identifica- dia in the shoot apex, in root tips, and in devel-
tion; about 100 are now known. oping fruits and seeds.
Most higher plants contain a number of There are no clear patterns of movement of
GAs although not all have intrinsic biological GA in the plant although they generally move
activity. In addition, GAs are also found in towards young, growing tissues in both xylem
fungi: GA3 and a mixture of GA4 and GA, from and phloem.
these sources are available commercially.
27.8.3 PHYSIOLOGICAL ACTIVITIES AND
APPLICATIONS OF GIBBERELLINS
27.8.1 BIOCHEMISTRY OF GIBBERELLINS
Effects on vegetative growth
GAs are diterpenoid derivatives and are syn-
thesized by the condensation of five-carbon (C- Application of GAs to intact plants often
5) units head-to-tail. Successive linking of these results in spectacular stimulation of shoot
units results in formation of geranyl pyrophos- elongation, but they generally have little effect
phate (C-10), farnesyl pyrophosphate (C-15) on elongation of isolated stem sections. They
and geranylgeranyl pyrophosphate (C-20) appear to stimulate cell division in the shoot
(Figure 27.7). Farnesyl pyrophosphate may also apex and to increase cell wall plasticity in stem
serve as a precursor for the synthesis of ABA cells. However the effects on wall plasticity do
(section 27.10.1) and steroids. Geranylgeranyl not seem to result from acidification of the
pyrophosphate undergoes cyclization to form wall, and synergistic interactions of gib-
ent-kaurene via copalyl pyrophosphate. Steps berellins and auxins in controlling elongation
up to this point are catalysed by soluble of cells are seen.
enzymes. Many dwarf plants have abnormalities in
The next steps take place on the endoplas- gibberellin biosynthesis or responsiveness.
mic reticulum and result in formation of GA12 The former types have been particularly use-
aldehyde, from which other GAs arise by steps ful in elucidating the pathways of gibberellin
which vary from genus to genus. biosynthesis whilst the latter form the basis of
The existence of mutants which are defec- many commercially important high yielding,
tive in GA synthesis has allowed the biosyn- semi-dwarf cereal varieties.
thesis of GA in shoots of pea and maize to be GA can be used to overcome some environ-
studied very thoroughly. GA l2-aldehyde is mental requirements for growth. Thus, GA
converted to GAl by a pathway called the early reduces the chilling requirement of rhubarb
ent-gibbereliane
GA 1
G~
Figure 27.6 The structure of the gibberellins is based on the ent-gibberellane ring. GAl is the biologically
active form in pea and maize shoots, and GA3, GA4 and GA7 are commonly used as growth regulators.
and increases the yield, replacing the need for increase yield. It may replace the need for long
forcing. It can also be used to overcome dor- days to induce flowering in rosette plants. GA
mancy in potato tubers. may also replace the need for exposure to low
temperatures (vernalization) to induce flower-
ing in some biennial or spring-flowering peren-
Effects on flowering
nials. GA has a number of indirect effects on
Young plants may show a juvenile growth flowering: for instance, it stimulates production
phase in which they will not flower, however of male flowers in cucumbers, leading to better
favourable the environmental conditions. pollination, and stimulates bolting in many
Application of GA may be used to reduce the rosette or biennial plants such as lettuce and
length of this unproductive juvenile phase of carrots, enhancing seed production. It has also
growth. proved very useful in the control of flowering
GA enhances flowering in many plants and in conifers as part of breeding programmes,
in some, such as the globe artichoke, this may reducing the time needed to produce seed.
Geranylgeranyl
3 x CH3 COSCoA pyrophosphate
~P)®
Acetyl CoA
inhibited by
1
CHrCH 20H CH3 1 AM01618. CCC
and phosphOn D
I
HO-C-CH 3
I
CHrC02H O-@® Copalyl
pyrophosphate
Mevalonic acid
inhibited by
phosphonD
1 CH 3 CH 3
A,,--~® ent-kaurene
Isopentenyl pyrophosphate
1 Kaunme oxidilse -
lrihiblted bY~.
CH 3 ·CH 3 pa~Df:1ind
~a-®® ~
Geranyl pyrophosphate
ent-kaurenoic
acid
ABA
1 .,/'
~®
CH 3 ·C02H
Famesyl pyrophosphate ~ STEROIDS
CH2 GA12
1 aldehyde
"" a-®®
Geranylgeranyl pyrophosphate
Figure 27.7 The biosynthesis of gibberellins from acetyl-CoA. Condensation of five-carbon units head-to-tailleads to production of geranyl-
geranyl pyrophosphate. Cyclization and oxidation result in production of GA12 aldehyde from which other GAs arise. Enzyme-catalysed reac-
tions in the second part of the pathway are inhibited by growth retardants.
Effects on fruit development Growth retardants inhibit the biosynthesis
of GA and many of their effects can be
Application of GA can overcome ineffective
reversed by simultaneous application of GA.
pollination in fruit trees such as plum, peach,
AM01618, CCC and phosphon 0 inhibit syn-
cherry, apple and pear, which flower early in
thesis of kaurene, whereas paclobutrazol,
the year and are particularly susceptible to
ancymidol and tetcyclasis inhibit kaurene oxi-
frost damage. GA may also be used to promote
dase (Figure 27.7). Although the general
fruit set in tomatoes and mandarin oranges. In
effects of all the growth retardants are similar,
some plants, combinations of GA with auxin
in that they reduce internode length in stems,
or GA with auxin and cytokinin may be more
particular retardants have advantages for spe-
effective than GA alone.
cific applications.
GA is often applied pre-bloom to open clus-
ters of grapes, giving greater separation. This
ensures that the berries dry more rapidly and Dwarfing of pot plants
that the fruit do not damage one another,
Application of growth retardants to pot plants
releasing juice and increasing susceptibility to
such as chrysanthemums, azaleas, poinsettias,
fungal infections.
carnations and geraniums produces bushier
plants with more flowers. They are very rapid-
Effects on seed dormancy and germination ly translocated and may be applied as a foliar
spray (except phosphonium chloride which
Treatment of seeds with GA can lead to more
may scorch leaves), as a soil drench, or incor-
uniform germination and hence better
porated into the soil.
seedling establishment, so that mechanical
harvesting of the crop becomes more effective.
It is widely used in malting as it stimulates ger- Lodging control in cereals
mination and a-amylase production in the
Lodging of cereals occurs in response to wind
grain, so that conversion of starch to fer-
and rain, particularly in fertile soils and when
mentable sugars is both accelerated and made
plants are weakened by eyespot infection.
more complete.
Application of CCC is widely used to reduce
lodging of wheat and oats even at high fertil-
Effects on cambial development izer application rates.
GA stimulates cambial growth and differentia-
tion in conjunction with auxins (Section 27.7.4). Correlative effects
Individual parts of a plant compete with one
27.8.4 GROWTH RETARDANTS
another for assimilates. Thus, yields of roots or
Growth retardants reduce the growth of fruits or seeds may often be increased by
plants by inhibiting the biosynthesis of GA. restricting vegetative growth by application of
They reduce the rate of stem elongation with- growth retardants. In addition, reduced
out reducing leaf number or leaf area. The growth of parts of no commercial value may
structures of some commonly used growth result in compensatory growth of more valu-
retardants are shown in Figure 27.8. able parts of the plant. Effects of this type are
More recently introduced growth retar- known as 'correlative effects'.
dants such as paclobutrazol and tetcyclasis are Application of daminozide, CCC or
effective at approximately one thousandth the paclobutrazol restricts the growth of apple
concentration at which older retardants, such and pear trees, producing more compact
as CCC, are normally used. plants which flower earlier. In young, vigor-
Gibberellins 383
CH 3
I+cr
CI-CHrCH r N-CH3
I
CH 3
CCC
Chlorocholine chloride Phosphon
Chlormequat chloride Chlorphonium chloride
Cycocel
o CH 3 NV
()-iD-°c~
II I
CH r C-NH-N
I I
CH 2-C02H CH 3
Daminozide Ancymidol
Alar A-Rest
89
Mepiquat chloride
Paclobutrazol
Figure 27.8 Structures of some commonly used growth retardants. Quaternary ammonium and phospho-
nium derivatives such as CCC and phosphon were amongst the first to be discovered. More recently
introduced compounds include paclobutrazol which is related to the triazole fungicides.
ous fruit trees, fruit drop may occur because of rise to better established plants with more fer-
competition for assimilates between the large tile tillers, with more heads and with the
number of rapidly growing fruits and the veg- potential to give higher yields.
etative growing points. This can be greatly
reduced by pinching out the shoot tips or by
Fruit quality
spraying with daminozide, which inhibits
shoot growth and diverts nutrients to the Daminozide (Alar) increases anthocyanin pro-
fruitlets. duction (and hence the red colour in apples),
Early application of growth retardants to decreases pre-harvest drop, and reduces soft-
cereals may reduce growth of the shoot apex, ening in cold conditions. It has therefore been
allowing development of additional tiller buds extensively used on red apples, especially in
and stimulating root development. This gives the USA. In 1989 there was considerable con-
cern because the compound was reported to cleavage of isopentenyl adenine are adenine
be weakly carcinogenic; despite this not being and 3-methyl-2-butenal [CHO-CH=C(CH3)2]
substantiated by an independent enquiry, the and all biological activity is lost, so this is a
compound was withdrawn from use for food means of inactivating cytokinins.
crops worldwide because of public reaction.
27.9.2 SITES OF SYNTHESIS AND TRANSPORT
OF CYTOKININS
27.9 CYTOKININS
In vegetative plants, the root tip seems to be
27.9.1 BIOCHEMISTRY OF CYTOKININS
the major site of cytokinin biosynthesis. These
Naturally occurring cytokinins are substituted compounds are also found in relatively large
adenine derivatives. Most are derivatives of amounts in young fruits, seeds and leaves and
zeatin or of N6-isopentenyladenine (Figure may be synthesized in some of these.
27.9). These compounds exist in the free form Cytokinins produced in the roots are trans-
as bases, their 5' -ribosides (zeatin riboside and ported in the xylem to the shoot. In other
isopentenyl adenosine, respectively), and 5'- respects their transport is very restricted, and
ribotides (zeatin ribotide and isopentenyl when applied exogenously they tend to
AMP). remain at their sites of application.
Cytokinins may be converted into gluco-
sides or amino acid conjugates. These are 27.9.3 PHYSIOLOGICAL ACTIVITIES AND
inactive but some may serve as a store from APPLICATIONS OF CYTOKININS
which active hormone can be regenerated by
Role in cell division and differentiation of
hydrolysis.
callus
Cytokinins also occur as components of
about 10% of tRNA molecules. When present, Callus cells replicate their chromosomes but
they always occur at the position immediately are unable to divide unless cytokinins are
next to the 3' end of the anticodon, and the added to the growth media. Stimulation of cell
tRNA molecules containing them all bind to division is the most characteristic effect of
mRNA codons beginning with U. these hormones. The effect of cytokinins on
Cytokinins are synthesized by transfer of an differentiation of callus is discussed in section
isopentenyl group from dimethyl allyl 27.7.4.
pyrophosphate to the N6-amino group of free Most plant cells growing in tissue culture
AMP or to an adenosine residue at the appro- require added hormones to grow rapidly, but
priate position in RNA. The enzyme which when cells become infected with the crown
catalyses this reaction has been characterized gall bacterium Agrobacterium tumefaciens they
(section 27.9.3). Zeatin derivatives are formed become hormone-autotrophic. The Ti
by hydroxylation of the isopentenyl side (tumour inducing) plasmid which becomes
chain. Some free cytokinins could arise from incorporated into the host-cell genome carries
breakdown of cytokinin-containing tRNA but genes coding for the biosynthesis of both
this does not appear to be an important auxin and cytokinins. As crown gall tissues
source. The physiological significance of contain much higher levels of hormones than
cytokinins in RNA and their relationship to normal tissues, they have been able to provide
free cytokinins is unclear. a great deal of information about auxin and
Some cytokinins can be oxidized by the cytokinin synthesis.
enzyme cytokinin oxidase, which catalyses Auxin biosynthesis in these galls requires
removal of the side chain from the N6-amino two enzymes - tryptophan-2-monooxygenase
group of the purine ring. The products of and indoleacetamide hydrolase, which catal-
Cytokinins 385
Zeatin N6-lsopentenyl adenine
Zeatin riboside N6-lsopentenyl adenosine
Zeatin ribotide N6 -lsopentenyl AMP
Figure 27.9 Structures of cytokinins. Naturally occurring cytokinins are mostly derivatives of zeatin and
isopentenyladenine. They exist in the free form as bases,S' -ribosides and 5' -ribotides.
yse the steps shown in Figure 27.10a. These ph ate with AMP to form isopentenyl-AMP
are coded for by the genes tmsl and tms2, (Figure 27.lOb).
respectively. Inactivation of tms genes coding for auxin
Two genes in A. tumefaciens code for biosynthetic enzymes results in greatly
enzymes which catalyse cytokinin synthesis. increased cytokinin/auxin ratios, leading to
One of these, tmr (also called ipt), codes for the crown galls which produce shoots, whilst inac-
enzyme DMAPP:AMP transferase which catal- tivation of genes coding for cytokinin biosyn-
yses the reaction of dimethylallyl pyrophos- thetic enzymes (tmr) results in decreased
(a)
Tryptophan
tryptophan-2-monooxygenase
1
W N
H
H:rC-NH2
II
o
Indole-3-acetamide
1
indo/eacetamide hydrolase
W><,-co,H Indole-3-acetic acid
(b)
AMP
CH 3
NH-CHrCH=b-cH 3
~j-) N6 -lsopentenyl AMP
~ibose-®
Figure 27.10 Pathways for the biosynthesis of (a) auxins and (b) cytokinins in Agrobacterium tumefaciens.
Bacterial genes tmsl and tms2 code for the auxin biosynthetic enzymes, and tmr for that which synthe-
sizes cytokinins. Infection of plant cells by Agrobacterium produces crown galls which contain much high-
er than normal levels of these hormones.
cytokinin/auxin ratios and crown galls which dence that a decrease in protein synthesis
produce roots. occurs during senescence. Although leaves
normally senesce quickly when detached from
the plant, this is prevented if they produce
Leaf development and senescence
roots or if a drop of cytokinin solution is
Cytokinins promote the growth of leaves and applied to the leaf. In the latter case the region
delay senescence. There is considerable evi- in contact with the cytokinin remains as a
Abscisic acid (ABA) 387
'green island' as the remainder of the leaf yel- ABA is very rapidly and extensively
lows. It is thus generally considered that root- metabolised in higher plants to form mainly
produced cytokinins delay leaf senescence by ABA-glucose ester and phaseic and dihy-
maintaining protein synthesis in leaves. drophaseic acids (Figure 27.11). Phaseic and
dihydrophaseic acids show little biological
activity and may be formed as a way of inacti-
Apical dominance
vating ABA.
Cytokinins promote development of lateral
buds in some species (section 27.7.4).
OF ABSCISIC ACID
Applications
Abscisic acid is synthesized in mature green
The principal synthetic cytokinins available leaves, especially in response to water stress. It
are benzyl adenine (BA) and kinetin. These are is also present in large quantities in seeds. ABA
used in combination with GA to improve fruit appears to move quite readily between the
set in grapes and figs, and to increase lateral leaves, shoots and roots.
bud growth and fruit quality in apples.
Although cytokinins have potential uses in
delaying senescence of green vegetables,
APPLICATIONS OF ABSCISIC ACID
mushrooms and cut flowers they are not wide-
ly used in this way.
Role in response to stress
When leaves are subjected to water stress the
27.10 ABSCISIC ACID (ABA)
rates of biosynthesis of ABA are greatly
The structure of abscisic acid is shown in increased. This is triggered by changes in turgor
Figure 27.11. Biological activity appears to be pressure which are detected by the plasma
associated with just this single compound. It is membrane. The elevated levels of ABA in the
a sesquiterpenoid containing 15 carbon atoms. leaves inhibit K+ accumulation in guard cells,
It is optically active, existing as +/- isomers at reducing their turgor and causing the stomata
the l' -position, and there are also cis/trans iso- to close, thus reducing water loss. When plants
mers about the 2-double bond. The naturally are re-watered, ABA concentrations drop to
occurring compound is ( +)-2-cis ABA. It occurs pre-stress levels within 4-8 hours, in this case
widely in angiosperms, gymnosperms, ferns due to decreased biosynthesis and rapid metab-
and mosses but a different compound, olism to phaseic and dihydrophaseic acids.
lunularic acid, may fulfil similar functions in The potential use of ABA as an antitranspi-
liverworts and some algae. rant is of considerable interest. Partial closing of
the stomata can decrease transpiration rates
without affecting photosynthesis, and can
27.10.1 BIOCHEMISTRY OF ABSCISIC ACID
enable plants to make more efficient use of
ABA is synthesized from mevalonic acid. The water. ABA itself is expensive and has short-
steps as far as the IS-carbon compound farne- lasting effects. Derivatives such as the methyl
syl pyrophosphate (FPP) are the same as those ester of ABA, however, can control stomatal
described for GA biosynthesis (Figure 27.7). opening for 9-10 days. Such compounds would
The conversion of FPP into ABA occurs via be of particular value for plants introduced into
another C-lS compound, xanthoxin. In higher climatically unsuitable, marginal areas especial-
plants it is likely that carotenoids are interme- ly at times when they are particularly suscepti-
diates in the conversion of FPP to xanthoxin. ble to water stress, such as during reproduction.
ABA
Phaseic acid Dihydrophaseic acid
Figure 27.11 The structures of abscisic acid (ABA) and metabolites. ABA is rapidly metabolized to form
phaseic acid which is then converted to dihydrophaseic acid.
Salinity and low temperature may also there is no correlation between ABA levels and
cause increased synthesis of ABA and this may depth of bud dormancy.
help the plant resist these stressful conditions Although ABA inhibits germination of the
(section 27.5) . seed of many species, and levels of endoge-
nous ABA fall when dormancy is broken in
some seeds, there is no universal correlation
Role in control of assimilate movement
between seed dormancy and ABA levels.
ABA, auxins and gibberellins appear to
increase assimilate transport from leaves into
27.11 ETHYLENE
developing seeds. ABA also activates genes
coding for specific storage proteins during Ethylene is unique amongst the hormones in
seed development and prevents premature being a gas and having a very simple structure
germination of the seed before they desiccate (Figure 27.12a). A number of synthetic com-
and mature. pounds decompose (either spontaneously or
through the action of enzymes) to produce
ethylene, and some other unsaturated hydro-
Role in inhibition of growth and in
carbons may mimic its effects.
dormancy of buds and seeds
ABA commonly inhibits the growth of cells
27.11.1 BIOCHEMISTRY OF ETHYLENE
and often counteracts the growth-promoting
activity of auxins, gibberellins and cytokinins. Ethylene is derived from carbon atoms 3 and 4
In some trees, levels of ABA in leaves and of methionine. Biosynthesis takes place via
buds increase in late summer when the buds amino cyclopropane carboxylic acid (ACC) as
become dormant, and levels decline as buds shown in Figure 27.12.
are chilled and dormancy is broken. ACC synthase [1] catalyses conversion of
Application of ABA can cause some buds to S-adenosyl methionine to ACC, which is con-
become dormant. In many species, however, verted to ethylene by the action of ACC oxi-
Ethylene 389
dase [2], formerly known as ethylene-form- High auxin concentrations usually inhibit
ing enzyme (EFE). As ACC oxidase requires plant growth, because they induce the produc-
oxygen, ACC accumulates under anaerobic tion of ethylene in the plant by increasing tran-
conditions. The activity of ACC oxidase scription of the gene coding for ACC synthase.
increases under some conditions which The biosynthesis of ethylene is reduced by
increase ethylene production such as stress, aminoethoxyvinylglycine (AVG) and amino-
in fruit ripening or in response to ethylene oxyacetic acid (AOA), which inhibit ACC
itself (autocatalysis). synthase by blocking the action of pyridoxal
phosphate. A single application of AVG may
prevent flowering in bromeliads for several
months. AOA has similar activity to AVG and
OF ETHYLENE
is considerably cheaper.
Most tissues can produce ethylene: ripening Ag+ and carbon dioxide are widely used to
fruit, the shoot apex, nodes of dicotyledonous reduce ethylene activity and to delay flower
seedlings, and flowers and leaves undergoing senescence and fruit ripening, as discussed
senescence are particularly rich sources. elsewhere in this chapter.
Ethylene production in most tissues is
increased in response to mechanical stresses
such as rubbing, pressure or damage or by
APPLICATIONS OF ETHYLENE
treatment with high concentrations of auxins.
Effects on fruit ripening
27.11.3 METHODS OF MODULATING THE There is much evidence that the ripening of
EFFECTS OF ETHYLENE ON PLANTS fruit is controlled principally by ethylene. In
most fleshy fruits the onset of ripening is pre-
Ethylene-induced responses can be manipu- ceded by a burst of ethylene production which
lated in several ways. stimulates respiration. Ethylene production is
Several small, unsaturated hydrocarbons an autocatalytic process; trace amounts lead to
and carbon monoxide have similar, but much production of increasing quantities.
weaker biological activity than ethylene (Table Ethylene may change membrane perme-
27.1). These compounds bind to the ethylene ability leading to the release of the enzymes
receptor which has relatively low specificity needed for ripening. It may also increase syn-
for ethylene. The receptor contains copper thesis of proteins, including those required for
and its function is inhibited by copper-chelat- hydrolysis of cell components and for the
ing agents such as ethylene diamine acetic
acid (EDTA), and by C02+ and Ag+. cO 2 acts an Table 27.1 Relative activity of ethylene analogues
antagonist and competes with ethylene for in inducing ethylene-like responses
binding to the receptor.
Compounds such as ethephon (Etherel) Structure Name Relative
effectiveness
and etacelasil (Figure 27.12) decompose in the
plant to produce ethylene. Silane derivatives CH 2 =CH2 Ethylene 1
such as etacelasil decompose more rapidly CH 3-CH=CH z Propylene 1/100
than ethephon and produce slightly different CH=CH Acetylene 1/2800
responses. CH 2 =C=CH 2 Allene 1/29000
When ACC is applied to plants it may be C=O Carbon monoxide 1/2700
converted into ethylene by the action of ACC
Data reproduced with permission from Burg, S.P. and
oxidase in an oxygen-requiring reaction (see Burg, E.A. (1967) Molecular requirements for the biologi-
Figure 27.12). cal activity of ethylene. Plant Physiology, 42, 144-152.
(a)
H
,C=C
/
H
H/ 'H
Ethylene
(b)
C02H C02H
I I
CH-NH2 CH-NH2
-
I
n
I
3 CH 2 CH2 [1]
I I
4 CH2 CH 2
I I
S S-Ado
I ATP PPi I
CH 3 + CH 3
Pi 1-aminocyclopropane 1-
Methionine S-adenosyl Ethylene
carboxylic acid
methionine
(ACC)
(c)
0
pH >4 H H
II
'C=C/ + OH-P-OH + cr
H/ 'H
OH
I
Ethephon Ethylene Phosphate
Etacelasil
Figure 27.12 The chemistry of ethylene and related compounds. (a) The structure of ethylene. (b) Pathway
for the biosynthesis of ethylene from methionine: ACC synthase catalyses reaction [1] and ACC oxidase
catalyses reaction [2]. (c) Reactions for the breakdown of ethephon and etacelasil to release ethylene.
increase in respiration. Both the biosynthesis or after harvesting, by applying ethephon to

of ethylene and the increased respiration trig- fruits such as apple, tomato, banana, melon,
gered by ethylene require oxygen. pepper and coffee.
Storage of fruit in air with a low oxygen The molecular biology of the ripening
content, a low ethylene content or a high car- process has been studied extensively because
bon dioxide content delays ripening. Low of the potential commercial value of fruits
temperature prevents ethylene production which ripen more slowly and have a longer
and reduces respiration rates. Conversely shelf life. Ripening may be delayed by reduc-
ripening can be promoted, either on the plant ing ethylene synthesis through the use of anti-
Ethylene 391
sense genes for ethylene biosynthetic vature of the leaves called epinasty. Ethylene
enzymes, or through the use of antisense promotes elongation of cells on the upper side
genes for enzymes, such as polygalacturonase of the petiole but not those on the lower side,
(PG) and pectinmethylesterase (PME), which resulting in curvature. Symptoms such as
are involved in ripening itself. The use of these may be seen in plants exposed to ethyl-
transgenic plants containing antisense genes ene or high concentrations of auxins, which
for ACC synthase or ACC oxidase has allowed stimulate ethylene production, or in plants
ethylene synthesis to be almost completely subjected to stress.
eliminated. Ethylene synthesis has also been Cells cannot extend parallel to the orienta-
reduced in transgenic plants containing the tion of the cellulose microfibrils in their cell
gene for ACC deaminase, which rapidly walls, but are able to do so perpendicular to
degrades ACC, reducing its availability for eth- them. In many stem and root cells, microfib-
ylene synthesis. Insertion of antisense genes rils are laid down predominantly with a hor-
for PG into tomatoes reduces the activity of izontal orientation, allowing the cells to
this enzyme and somewhat delays the soften- extend vertically. Exposure to ethylene
ing which accompanies ripening. Transgenic changes the orientation, reducing vertical
fruit of this type are marketed by Calgene and promoting horizontal growth. This is
under the trade name FlavrSavr®. important in the emergence of seedlings
where thickening of the stem and root gives
Promotion of abscission of leaves and fruits them greater strength and enables them to
push through compacted soils.
Ethylene promotes abscission of both leaves Ethylene synthesis can be stimulated by
and fruits. Abscission occurs when cells in a stresses such as rubbing or cutting stems and
separation layer synthesize pectinase and spe- leaves, attack by pathogens or exposure to
cific isozyme forms of cellulase in response to
drought or flooding. In some plants, ethylene
ethylene. These enzymes degrade cell walls
produced on wounding initiates secretory
and cause the cells to part.
processes. A commercially important exam-
Where mechanical harvesting is employed,
ple is the stimulation, by application of ethep-
ethylene-generating compounds may be used
hon, of latex flow in rubber trees following
to remove leaves in order to facilitate harvest-
tapping. A 10% solution of ethephon in palm
ing of seeds or fruits such as cotton and some
types of beans. Fruit harvesting may also be oil applied to the bark of the tree just below
facilitated by treatment with a 'harvest-aid' the normal cut increases latex and dry rubber
which reduces the force required to remove production by up to 100%. Ethephon appears
the fruit, thus reducing mechanical damage to to prevent the vessels from becoming
both fruit and tree. Ethephon is extremely blocked.
effective when used on apples, cherries, citrus Ethylene diffusion through water is slow
fruits, olives, pears and grapes. However other and it cannot readily escape into waterlogged
ethylene-generating compounds such as soils. In addition, roots become anaerobic,
etacelasil, which is commonly used in olives, reducing ethylene formation from ACC (sec-
cause less leaf abscission. tion 27.11.1). Under these conditions ACC
moves to the shoot where, in the aerobic envi-
ronment, it is rapidly converted into ethylene
Effects on stem elongation and leaf and is responsible for many of the characteris-
development tic symptoms of waterlogging. Some types of
Exposure of pea seedlings to ethylene reduces rice are unusual because ethylene promotes
stem elongation and causes a downward cur- elongation of their internodes.
Effects on flowering and flower senescence and removal of ethylene from the atmosphere
are all beneficial.
Ethylene-producing chemicals are used to
promote flowering in mangoes and bromeli-
ads (section 27.7.4). 27.12 MISCELLANEOUS PLANT GROWTH
Ethylene production, particularly by the REGULATORS
stigma and style, decreases the storage life of
27.12.1 MORPHACTINS
cut flowers by promoting senescence. Auxin in
pollen may also stimulate ethylene production The morphactins such as chlorflurecol are a
after fertilization. Some species such as carna- group of compounds which are derivatives of
tions are particularly susceptible to ethylene. fluorene-9-carboxylic acid. They disrupt coordi-
The storage life of cut flowers can be nated growth and reduce apical dominance,
increased by a process called pulsing, in possibly by affecting basipetal auxin transport.
which they are immersed in solutions con- Chlorflurecol methyl is used in combination
taining a mixture of sucrose, a biocide, a weak with maleic hydrazide to delay growth of grass-
acid, an anti-ethylene agent and sometimes a es and broad-leaved weeds on roadside verges.
growth regulator. Sucrose replaces carbohy-
drates which are depleted by respiration
27.12.2 MALEIC HYDRAZIDE
occurring during storage. It also decreases
ethylene production and increases the ability Maleic hydrazide has been used for over 40
of plant tissues to retain water. Biocides such years and was, at one time, the most com-
as silver nitrate, 8-hydroxy quinoline and alu- monly used plant-growth regulator. It is a
minium sulphate prevent growth of bacteria growth inhibitor, restricting cell division in
which may plug the stem or produce toxic both the apex and sub-apical region. It there-
metabolites and ethylene. Silver nitrate is an fore reduces not only stem elongation, but
anti-ethylene agent as well as an effective bac- also the initiation of leaves and flowers. It is
teriocide. Silver thiosulphate is less effective used to control growth of suckers in tobacco,
as a biocide but is very effective indeed in and also at the bases of amenity trees, to pre-
inhibiting both the production and action of vent sprout growth in potato tubers and
ethylene. It may double the life of those flow- onions, and to reduce the growth of hedges
ers, such as carnations, which are particularly and grasses on verges.
sensitive to ethylene. Treatment of cut flowers
with cytokinins improves keeping qualities
27.12.3 GLYPHOSINE
but is rarely more effective than silver thiosul-
phate treatment. Glyphosine is used as a sugarcane-ripening
The manipulation of the atmosphere in agent. It inhibits the late stages of sugarcane
which flowers are stored can be beneficial to growth, causing sucrose to be stored in the
their longevity in much the same way as for stem instead of being used for fibre produc-
fruits. Thus lowering temperature and raising tion, and leads to increases in yield of sucrose
CO 2 concentration, reducing 02 concentration of 10-15%.
PART FOUR
STRATEGIES FOR PROCESSING OF NUTRIENTS

IN ANIMALS
DIGESTION AND ABSORPTION IN 28
RUMINANTS AND NON-RUMINANTS

28.2 Structure of the digestive tract 395
28.3 Carbohydrate digestion in monogastric 396
animals
28.4 Carbohydrate digestion in ruminants 398
28.5 Absorption and utilization of glucose 398
28.6 Digestion of lipids in monogastric 399
animals
28.7 Absorption of lipid from the small 402
intestine
28.7.1 Glycerides and fatty acids 402
28.7.2 Phospholipids 403
28.7.3 Cholesterol 403
28.7.4 Chylomicron formation 403
28.8 Uptake of absorbed lipid by body tissue 403
28.9 Digestion of lipids in ruminant animals 404
28.10 Lipid digestion in poultry 406
28.11 Digestion of proteins in monogastric 406
animals
28.12 Digestion of protein in ruminants 409
28.13 Inhibitors of digestive enzymes 410
28.1 INTRODUCTION This is followed by absorption of digestive

Animals consume food consisting of a com- end products across the wall of the different
plex matrix containing both simple molecules compartments of the digestive tract, and their
(free sugars and amino acids, etc.), and macro- distribution to body tissues via the blood
molecules such as structural carbohydrates, stream. Digestion is a physicochemical
protein and lipids. For the most part, simple process in which enzymes, secreted both by
molecules require little modification before the digestive tract and by microorganisms
they can be absorbed from the digestive tract present within it, are utilized to catalyse the
and utilized by the tissues. However, macro- breakdown of complex molecules. The end
molecules almost always require dismantling products of digestion vary from species to
into their simpler building blocks before they species. The nature of these products is influ-
can be absorbed across the intestinal wall. enced by the extent to which food is degrad-
Digestion is the process by which these ed by microbial fermentation or endogenous-
dietary macromolecules are broken down. ly synthesized enzymes.
396 Digestion and absorption in ruminants and non-ruminants
28.2 STRUCTURE OF THE DIGESTIVE TRACT ,:h.ich is capable of cleaving the a-1,4 glyco-
SIdIC bonds between the glucose residues of
A~im~ls have developed a wide variety of
amylose, amylopectin and glycogen. This
dIgestIve strategies to cope with the great vari-
ation in the nature and digestibility of the enzyme (which has a pH optimum of 7.0) is
identi~al to pancreatic amylase, but normally
feedstuffs they consume. This is reflected in
the diversity of digestive anatomy seen in the has httle effect on dietary carbohydrate
because of the relatively short time it is in con-
animal kingdom. Among the monogastric
tact with food before being inactivated by the
species there are considerable differences in
acidic conditions of the stomach.
the size of the digestive tract, and in the con-
Pancreatic a-amylase and l3-amylase are the
tribution made by each of the three main com-
two enzymes mainly responsible for degrada-
partments of the digestive tract, the stomach,
tion of a-linked carbohydrates in the small
small intestine and hindgut (caecum and
intestine. They are secreted in the pancreatic
colon), to the process of digestion.
juices and mixed with digesta in the proximal
In carnivorous animals such as feline and
duodenum. a-Amylase is an endoglycosidase
canine species, the digestive tract is relatively
which catalyses the random hydrolysis of a-
short and simple. Digestion occurs mainly
1,4 glycosidic bonds between glucose residues
within the stomach and small intestine
(Figure 28.1). I3-Amylase is an exoglycosidase
through the action of endogenous enzymes.
which removes glucose residues from the non-
The hindgut, where fermentation takes place,
reducing ends of glucose chains. The linear
IS poorly developed. In contrast, monogastric
chains of amylose are degraded to a mixture of
herbivores such as the rabbit and horse are
glucose and the disaccharide maltose.
dependent not only on enzymic digestion in
Amylopectin and glycogen are branched
the stomach and small intestine, but also on
str~ctures (Chapter 3) in which glucose
the subsequent microbial fermentation of the
reSIdues at the branch points are linked by a-
structural carbohydrates from forages in the
1,6 glycosidic bonds that are not hydrolysed
caecum and/or colon. Omnivores such as the
by amylases. The end product of amylopectin
pig fall between these two extremes.
and glycogen degradation by amylase is a
Ruminants differ from monogastric herbi-
highly branched structure known as limit dex-
vores in digestive anatomy. They are reliant
trin. This is degraded by the action of
on pregastric fermentation of dietary con-
debranching enzyme to produce numerous
stituents in the rumen. Fermented food passes
short, linear chains of glucose that are then
from this large fermentation chamber through
further degraded to maltose and glucose by
the omasum and into the abomasum, which is
amylases. Maltose is hydrolysed to glucose by
the true glandular stomach of ruminant
maltase, one of a number of disaccharidases
species. The importance of the rumen is
associated with the brush border of the small
reflected in the fact that it represents between
intestine. Other disaccharidases are sucrase
60 and 70% of the volume of the digestive
which converts sucrose to glucose and fruc~
tract. The relative sizes of the digestive com-
tose; and lactase, which hydrolyses lactose to
partments in monogastric and ruminant
glucose and galactose.
species are shown in Table 28.1.
Non-starch polysaccharides in roughages
and cereals contain a variety of sugars
28.3 CARBOHYDRATE DIGESTION IN (Chapter 3). Cellulose is a homopolymer of glu-
MONOGASTRIC ANIMALS cose residues linked by 13-1,4 glycosidic bonds
which are not hydrolysed by enzymes secreted
In monogastric animals, little digestion of into the small intestine. Hemicelluloses contain
dietary carbohydrate occurs before it reaches a mixture of arabinose, xylose, mannose, galac-
the small intestine. Saliva contains an amylase tose and glucuronic acid, linked by a variety of
Carbohydrate digestion in monogastric animals 397
Table 28.1 Volume of main compartments of digestive tract
expressed as a percentage of the total volume of the tract
Compartment of digestive tract

Animal Rumen Stomach Small intestine Caecum Colon
Dog 62 23 14
Pig 29 33 8 30
Rabbit 15 12 23 50
Horse 9 21 16 54
Sheep 60 8 19 3 10
Cow 67 5 18 3 7
glycosidic bonds which are resistant to amylase volatile fatty acids: acetate, propionate and
hydrolysis. Typical hemicelluloses, xylans, con- butyrate. In monogastric herbivores the
tain a core polymer of [3-1,4 xylose and a num- hindgut is greatly enlarged to allow time for
ber of branches with a variety of other sugars. microbial fermentation of these carbohydrates.
Glucose-containing hemicelluloses, [3-glucans, The volatile fatty acids are absorbed across the
are typical of plant seeds. These hemicelluloses intestinal wall and are a valuable source of
are known as the cereal gums. They constitute energy. Measurements in the pig suggest that
a major proportion (70-75%) of the carbohy- volatile fatty acids absorbed from the hindgut
drate content of the endosperm cell wall, but can contribute as much as 1.2 MJ kg-D75 live
overall are a minor component of the grain. weight compared with 2.5-3.3 MJ kgD·75 from
These non-starch polysaccharides pass to VFA absorbed from the rumen in cattle and
the large intestine where they undergo degra- sheep. (See Chapter 30 for an explanation of
dation by microbial fermentation. The path- metabolic body weight, kgD75.)
ways of fermentation are discussed in Chapter There is considerable interest in the use of
16. The end products of the fermentation microbial enzymes, now produced on a com-
process are methane, carbon dioxide, and the mercial scale, to improve the digestibility of
Figure 28.1 Site of action of a-amylase, f3-amylase and debranching enzyme on starch and glycogen.
cereal non-starch polysaccharides in pigs and trast, when finely ground cereals are eaten, the
poultry. These carbohydrates increase the vis- small starch particles may pass rapidly
cosity of the digesta in the small and large through the rumen before fermentation of the
intestine, resulting in depressed digestibilities starch is complete. For this reason cereals are
of other nutrients and reduced growth perfor- often rolled, bruised or flaked to rupture the
mance. The use of carbohydrase enzyme sup- pericarp and expose the starch granules with-
plements has been shown to reduce digesta out markedly reducing particle size.
viscosity, improve mixing of digesta and Non-starch polysaccharides, not degraded
increase growth performance, presumably in the rumen, pass through the small intestine
through enhanced nutrient absorption. and undergo further fermentation in the
hindgut; however, this makes only a small
contribution to the total VFA production in the
28.4 CARBOHYDRATE DIGESTION IN
gut.
RUMINANTS
The digestion of carbohydrates in ruminants
28.5 ABSORPTION AND UTILIZATION OF
occurs mainly in the rumen. Rumen microor-
GLUCOSE
ganisms produce a variety of enzymes capable
of degrading starch, cellulose and other non- The absorption of glucose fn?m the small intes-
starch polysaccharides. Starch is rapidly tine occurs via active transport. The apical
degraded to glucose by the action of bacterial membrane of the mucosal cells lining the
amylases. Cellulose is more slowly degraded, lumen of the digestive tract is highly special-
by the action of 13-1,4-glucosidases, to cel- ized for the absorption of nutrients The sur-
lobiose which is then split into glucose. face area of this membrane is extremely large
Hemicelluloses are degraded to xylose-rich due to the presence of numerous microvilli.
oligosaccharides and then to monosaccharides. The disaccharidases which act upon maltose,
The free sugars, mainly glucose, are rapidly sucrose and lactose are intimately associated
metabolized to pyruvate by the glycolytic with the extracellular surface of these
pathway and are then converted to the volatile microvilli. The monosaccharides produced are
fatty acids acetate, propionate and butyrate by passed to a Na+ -dependent carrier protein
the pathways described in Chapter 16. The that transports the sugars into the cell by uti-
nature of the carbohydrate in the diet has a lizing the difference in the Na+ concentration
marked influence on the molar proportions of of the digesta and mucosal cell cytoplasm. Na+
the volatile fatty acids produced. Forage diets is transported into the mucosal cells down a
produce a predominantly acetate fermenta- concentration gradient and 'pulls' sugars into
tion. The introduction of cereals into the diet the mucosal cell against a concentration gradi-
results in a shift towards the production of ent. A low intracellular Na+ concentration is
larger quantities of propionate (Table 28.2). maintained by a Na+/K+ ATPase in the basal
Some starch may escape fermentation in membrane of the cell, which removes excess
the rumen and pass into the small intestine Na+ to the extracellular fluid in exchange for
where it is digested to glucose by amylases. K+ (Figure 28.2).
The type and degree of processing of cereals Absorbed sugars enter the portal circulation
influences the amount of starch which may and pass through the liver, which is able to
reach the small intestine. When whole cereal metabolize a variety of them for metabolic
grains are consumed, the presence of the purposes. For instance, galactose and fructose
intact pericarp may prevent access of the feed into the glycolytic pathway or can be con-
microbial enzymes to starch granules which verted to glucose. Glucose may be incorporat-
pass undegraded to the small intestine. In con- ed into glycogen or metabolized to provide
Digestion of lipids in monogastric animals 399
Table 28.2 Molar proportions of VFA in rumen fluid from
sheep fed differing proportions of hay and concentrates
Molar proportions of volatile fatty acids

Hay:concentrate ratio Acetate Propionate Butyrate
0.8:0.2 0.61 0.22 0.09

0.6:0.4 0.61 0.25 0.11
0.4:0.6 0.52 0.34 0.12
0.2:0.8 0.40 0.40 0.15
Adapted from: McDonald, P., Edwards, R.A., Greenhalgh, J.F.D. and

Morgan, c.A. (1995) Animal Nutrition, 5th edn, Longman Scientific
and Technical, Harlow, Essex.
energy and the precursors for the synthesis of glycogen in tissue reserves and the synthesis
amino acids and lipids. Glucose in excess of of glucose via gluconeogenesis. The plasma
hepatic requirements passes to the peripheral concentrations of insulin and glucagon change
tissues. In muscle, the main fate of glucose is in a reciprocal manner. Changes in the ratio of
oxidation to provide energy or storage as their plasma concentrations reflect changes in
glycogen. In adipose tissue, glucose may be the plasma glucose concentration.
used to provide acetyl-CoA for fatty acid syn-
thesis and glycerol-3-phosphate for triacyl-
28.6 DIGESTION OF LIPIDS IN
glycerol synthesis. The mammary gland has a
MONOGASTRIC ANIMALS
very high glucose demand for energy and for
lactose, fatty acid and triacylglycerol synthesis The main types of lipids found in feedstuffs are
(see Chapter 31). The brain and nervous tissue triacylglycerols, the storage lipids of plants and
are largely dependent on glucose as a supply animal tissues; glycolipids and phospholipids,
of energy, and this is the main reason for the the structural lipids of plant and animal mem-
very tight regulation of plasma glucose con- branes; cholesterol and cholesterol esters. The
centrations in animals. In man, the nervous relative proportions of these types of lipids
system accounts for about 70-80% of the total vary with the nature of the food consumed.
body glucose requirement in the resting state.
The nervous system is a much smaller propor-
28.6.1 DIGESTION IN THE STOMACH
tion of body weight in farm animals, and
therefore places a lower demand on the avail- The main site of lipid digestion is the small
able glucose. In the sheep, which is adapted to intestine; however, minor modifications to
lower plasma glucose concentrations, this may dietary lipid take place before the food reaches
account for only 10-15% of total body glucose. the intestine. A lipase has been detected in sali-
Plasma glucose concentrations are regulat- va and in the stomach, and these two enzymes
ed by the hormones insulin and glucagon. appear to be identical. They have a pH opti-
When plasma glucose concentrations rise, for mum of 4 and thus retain activity in the acidic
example after ingestion of a starch-rich diet, gastric environment. In adult animals they are
the pancreas secretes insulin which increases of limited importance, but in suckling animals
the uptake of glucose by the tissues and pro- they may make a significant contribution to the
motes glycogen synthesis. Glucagon secretion digestion of milk fat. The limited information
by the pancreas is stimulated by a fall in plas- available on these enzymes suggests that they
ma glucose, and promotes the breakdown of preferentially hydrolyse the fatty acid esteri-
lLmen of intestine
)
8
~t::\
'C7~
Blood
Figure 28.2 Mechanism of glucose absorption from the small intestine.
fied to the 3 position of the triacylglycerol and nents: bile, which contains bile salts, is required
exhibit specificity for short- and medium-chain for the emulsification of lipid contained in
fatty acids. This further supports the sugges- digesta leaving the stomach; and pancreatic
tion that they are most important in suckling juice which contributes the enzymes pancreat-
mammals as milk fat is unusual in containing ic lipase, phospholipase (AI and A2) and cho-
relatively high proportions of short- and medi- lesterol esterase, and the non-enzymic protein
um-chain fatty acids. The stomach also con- co-lipase. On entering the small intestine,
tributes to lipid digestion by denaturation of digesta is mixed with pancreatic juice which
the protein component of ingested lipopro- contains bicarbonate. This neutralizes the
teins, and by the production of an oil-in-water acidic digesta leaving the stomach. Typical pH
emulsion due to the vigorous mixing of the values for the gut contents of the pig are 2.4
stomach contents. (stomach); 6.1 (proximal duodenum); 6.8 (distal
duodenum); 7.4 Oejunum) and 7.5 (ileum).
Pancreatic lipase is responsible for the
28.6.2 DIGESTION IN THE SMALL INTESTINE
release of fatty acids from triacylglycerols. The
In the small intestine, efficient lipid digestion enzyme is most active between pH 6 and 7,
requires the presence of two main compo- and preferentially hydrolyses the fatty acids
Digestion of lipids in monogastric animals 401
esterified to the 1 and 3 positions of the tri- glycochendeoxycholate. The ratio of taurine-
acylglycerol molecule. 2-Monoacylglycerols esterified bile salts to glycine-esterified bile
and free fatty acids are the main end products salts is about 1:3 in monogastrics and 2.5:1 in
of lipase action. A small amount of the 2- ruminants. The detergent effects of bile salts
monoacylglycerols may be further digested by stem from their amphiphilic properties. The
the action of isomerases, which catalyse migra- hydrocarbon ring structure of these molecules
tion of the fatty acid from the 2 position to the is planar. The polar functional groups are
1 position to produce 1-monoacylglycerols, located on one surface of the molecule while
which are promptly hydrolysed by pancreatic the opposite surface is non-polar; thus they
lipase. Between a quarter to a third of dietary are able to coat the surface of fat droplets and
triacylglycerols are completely degraded to provide each droplet with a polar surface
glycerol and free fatty acids (Figure 28.3). which prevents the droplets from coalescing.
Pancreatic lipase acts at the surface of fat The bile salt layer surrounding the fat
droplets in the digesta. The rate of lipolysis is droplets is a barrier preventing the direct
dependent on the surface area of the droplets. attachment of pancreatic lipase. However, co-
Bile salts have a detergent action, breaking the lipase, a small protein secreted by the pan-
large fat droplets into numerous smaller ones, creas, is able to penetrate the bile salt layer and
thereby increasing the surface area available provides anchor points on the droplet to
for attachment of pancreatic lipase. The main which the lipase can attach (Figure 28.4).
types of bile salts found in bile are tauro- As digestion progresses, the products of tri-
cholate, glycocholate, taurodeoxycholate, gly- acylglycerol breakdown are transferred from
co deoxycholate, taurochendeoxycholate and the bulk fat phase to structures called micelles.
o
o "
CHp-C-Rl
" I
R2-C-o-9H
CH2"H
~
o
o CHp-e-R 1 o CH~H
/
" I " I
R2-C-o-CH 0 R2-C-O-9H +
I "
CHp-C-R3 • CH2"0H
2-monoacylglycerol
Triacylglycerol
oII CH-OH
I 2
R2-C-o-CH 0
CH-O-C-R
2 3
mixed 1,2 and 2,3-diacylglycerols
o
o
"
CH2"H
I
CH2"-C-R1
I 9
H2-OH
R2-C-O-?H -----+- HO-CH + R1COOH
- - - - - - HO-CH
I I
CH2'lH CH~H CH 2-oH
2-monoacylglycerol 1-monoacylglycerol glycerol

Figure 28.3 Breakdown of triacylglycerols in the small intestine.
salts
Pancreatic _ _ _ _•
lipase ~---Fat droplet
Colipase
Figure 28.4 Attachment of pancreatic lipase to fat droplets in the small intestine.
These structures are roughly spherical in 28.7 ABSORPTION OF LIPID FROM THE
shape and consist of an outer coating of bile SMALL INTESTINE
salts and phospholipid (both undigested phos-
The end products of lipid digestion are
pholipid and lysophospholipids) surrounding
absorbed across the intestinal wall in the
a core of undigested triacylglycerols, together
jejunum and proximal ileum. The mechanism
with the polar digestion products fatty acids
of transfer of micellar contents from mixed
and 2-monoacylglycerols, which are orientated
micelles is poorly understood, but it is clear
with their polar functional groups towards the that bile salts do not enter the intestinal
surface of the micelle (Figure 28.5).
mucosa in this region of the small intestine.
Because these micelles contain a mixture of
They are absorbed in more distal regions of the
the products of digestion (tri-, di-, and 2- ileum and recycled via the liver. This consti-
monoacylglycerols and fatty acids) they are tutes the enterohepatic circulation of bile salts.
referred to as mixed micelles.
Phospholipid is digested by the action of
phospholipases Al and A2 (see Figure 13.13)
28.7.1 GLYCERIDES
yielding 2-acyl lysophospholipids and I-acyl
lysophospholipids, respectively. The distribu- The detailed mechanism of uptake of fatty
tion of polar groups in these molecules gives acids and 2-monoacylglycerols into the intesti-
them amphiphilic properties which enables nal mucosa is unknown. It has been generally
them to work in concert with the bile salts at accepted that uptake is by passive diffusion,
the surface of the fat droplets and enhances but recent work suggests that membrane-
solubilization of the digesta lipid phase. mediated processes involving fatty acid-bind-
Fatty acids
+ - - - Bile salts
Figure 28.5 Representation of a mixed micelle.

Uptake of absorbed lipid by body tissue 403
ing proteins may be involved. In the intestinal are relatively large, between 50 and 200 J.Lm in
mucosa a process of resynthesis of lipid occurs. diameter, and consist of a hydrophobic lipid
Both triacylglycerols and phospholipids are core containing triacylglycerols, cholesterol
synthesized. The synthesis of triacylglycerols and cholesterol esters surrounded by a protein
occurs mainly by the 2-monoacylglycerol and phospholipid coat. Their average compo-
pathway, a pathway unique to the intestinal sition is 85% triacylglycerol, 9% phospholipid,
mucosa (Chapter 19). Absorbed fatty acids are 4% cholesterol and cholesterol esters and 2%
activated to their coenzyme A derivatives and protein, mainly apolipoproteins B-48, C-II and
are then esterified to 2-monoacylglycerols by C-III. The synthesis of chylomicrons occurs on
the sequential action of monoacylglycerol the mucosal cell Golgi bodies. The nascent
acyltransferase and diacylglycerol acyltrans- chylomicrons are transported to the basal
ferase. There may also be limited synthesis of membrane of the mucosal cells and are then
triacylglycerols by the glycerol-3-phosphate released into the lymphatic system.
pathway (Chapter 19). The role of fatty acid-
binding proteins in the process of resynthesis
28.8 UPTAKE OF ABSORBED LIPID BY BODY
remains unclear. These proteins are present in
TISSUE
significant quantities in the mucosal cells, but
it is not known whether they merely act as a The lymphatic system draining the digestive
vehicle for the transport of absorbed fatty tract empties via the thoracic duct into the sub-
acids, or whether they playa more important clavian vein. Chylomicrons entering the blood
role in the regulation of lipid synthesis. stream are rapidly transported around the
body, where the lipid contained within them
28.7.2 PHOSPHOLIPIDS can be utilized by the tissues. The process of
lipid uptake by tissues is mediated by the
The major substrates for phospholipid synthe- enzyme lipoprotein lipase, which is widely
sis are the lysophospholipids absorbed from the distributed throughout the body and is associ-
intestinal lumen. These molecules undergo re- ated with the endothelial cells of the capillar-
acylation by lysophosphotidate acyltransferase ies. The presence of chylomicrons containing
on the mucosal endoplasmic reticulum. Some apolipoprotein C-II activates the enzyme
de novo synthesis of phospholipid also occurs in which catalyses the hydrolysis of triacylglyc-
the mucosa via the glycerol-3-phosphate path- erols to glycerol and free fatty acids in the
way. The main end product of this pathway is lumen of the capillaries. The released fatty
phosphatidylcholine. acids are absorbed across the capillary wall
into the underlying tissue (Figure 28.6).
28.7.3 CHOLESTEROL The fate of absorbed fatty acids is deter-
mined by the function of the tissue into which
Cholesterol absorption from the intestinal they are absorbed. In adipose tissue, they are
lumen is an inefficient process. Much of the reincorporated into triacylglycerols and stored
absorbed cholesterol is re-esterified during for future use. In muscle and other tissues,
passage through the mucosal cell, probably by they may undergo oxidation to provide ener-
a reversal of the cholesterol esterase reaction. gy or be used for structural lipids. In liver, they
may be oxidized, converted to structural
lipids, or incorporated into triacylglycerols
28.7.4 CHYLOMICRON FORMATION
and phospholipids prior to inclusion in very
Resynthesized lipids are incorporated into low density lipoproteins (VLDLs) which are
chylomicrons for transport to the blood secreted into the blood stream for use by other
stream. This type of lipoprotein is associated tissues in the body. In mammary tissue,
exclusively with the intestinal mucosa. They lipoprotein lipase provides the mechanism for
Triacylglycerols
Figure 28.6 Tissue uptake of fatty acid via the action of lipoprotein lipase.
the uptake of circulating fatty acid of both tion. Also present are geometrical (trans) and
dietary and endogenous origin for use in the positional isomers of unsaturated fatty acids
synthesis of milk fat. (intermediates in the biohydrogenation
process). Fatty acids passing out of the rumen
are bound to the surface of feed particles. It is
28.9 DIGESTION OF LIPIDS IN RUMINANT
somewhat paradoxical then that in the grazing
ANIMALS
ruminant, whose diet is particularly rich in
The digestion and absorption of fatty acids is polyunsaturated fatty acids (a9,12-C18:2 and
different in ruminants from that in non-rumi- a9,12,15_C18:3), the fatty acids entering the small
nants, because the rumen microflora has a pro- intestine are predominantly saturated in
found effect on the nature of the lipid reaching nature.
the small intestine. The main types of lipids Lipid added to ruminant diets can be pro-
present in a ruminant diet are galactolipids tected against the effects of rumen microor-
(the main dietary lipid of a grazing ruminant), ganisms. The most effective form of protection
triacylglycerols and phospholipids. Rumen is achieved by coating fine droplets of lipid
microorganisms secrete lipases which very with casein which is treated with formalde-
rapidly hydrolyse dietary lipids to free fatty hyde to cross-link the protein molecules. This
acids, glycerol, sugars and phospholipid bases. renders the protein undegradable by the
The glycerol and sugars are quickly fermented rumen microflora and prevents enzymic
to volatile fatty acids. Fatty acids are further breakdown of the encapsulated lipid. When
modified by biohydrogenation in which most the protected lipid enters the abomasum (the
of the unsaturated fatty acids are converted to gastric stomach), the acidic conditions cause
their respective saturated derivatives (Chapter denaturation of the casein coating and the
16). The consequence of this metabolism is that lipid is released to be digested in the small
lipid leaving the rumen is composed almost intestine. This type of protection offers several
entirely of saturated fatty acids and smaller benefits. Firstly, it prevents biohydrogenation,
proportions of dietary unsaturated fatty acids allowing unsaturated fatty acids to pass to the
which have escaped complete biohydrogena- small intestine unaltered and thus offering the
Digestion of lipids in ruminant animals 405
potential to manipulate the fatty acid compo- 4.7-7.6 (distal jejunum) and 8.0 (ileum) (see
sition of ruminant tissues and products. comparable figures for the pig in section
Secondly, it protects the rumen microflora 28.6.2). The rate of transfer of fatty acids from
from the adverse effects of high levels of lipid feed particles to micelles increases as digesta
in the diet. Under normal conditions dietary passes from the duodenum into the jejunum.
lipid should not exceed 5-7% of the dry matter This process is aided by the presence of bile
content of the diet, as above this level dietary phospholipids. The amphiphilic nature of the
lipid, particularly if rich in unsaturated fatty phospholipids, and the lysophospholipids
acids, depresses rumen fibre digestion. produced from them by pancreatic phospho-
Protection of dietary lipids prevents these lipase Al and A2, enhances the solubilizing
adverse affects and allows the energy density effect of the bile salts. Absorption of fatty acids
of ruminant diets to be increased. This is par- from micelles occurs in the jejunum and is vir-
ticularly important in high-yielding dairy tually complete by the time the digesta reach-
cows (see Chapter 31). Alternative strategies to es the ileum.
the use of casein-protected fats have been Pancreatic lipase is present in ruminant
developed, such as the feeding of the calcium pancreatic juice, but is effectively redundant
salts of fatty acids and high-melting-point fats, in a grazing ruminant. In intensively fed rumi-
which are insoluble in the rumen and there- nants, where the proportion of triacylglycerol
fore do not adversely affect fibre digestion. in the diet increases, small amounts of lipid
However, biohydrogenation is not prevented may escape digestion in the rumen and pro-
completely by the feeding of these insoluble vide a substrate for the enzyme. In animals fed
fat sources. protected fat, dietary triacylglycerol enters the
Enzymic lipid digestion in the small intes- duodenum unaltered and is digested by the
tine of a ruminant fed unprotected fat is min- action of pancreatic lipase in a similar manner
imal as the bulk of the dietary lipid has to that in monogastric animals. Under these
already been digested to free fatty acids in the circumstances the end products of digestion
rumen. The most important function of the are a mixture free fatty acids and 2-monoacyl-
small intestine is release of fatty acids from glycerols.
the surface of feed particles and their incorpo- It is assumed that the mechanism of uptake
ration into micelles prior to absorption. This is of fatty acids from the ruminant digestive tract
achieved by the secretion of bile containing into the intestinal mucosa is similar to that in
bile salts and phospholipids (principally phos- monogastric animals. However, the pathway
phatidylcholine). Ruminant bile is particularly of triacylglycerol resynthesis in the mucosal
rich in taurine-conjugated bile salts, which are cells of animals fed unprotected fat differs
soluble and partially ionized at pH 2.5 (com- from that in monogastric animals, as there is a
pared with the glycine-conjugated bile salts, virtual absence of 2-monoacylglycerol.
the largest constituent of monogastric bile Instead, triacylglycerol synthesis occurs by the
salts, which are insoluble below pH 4.5). This glycerol-3-phosphate pathway (Chapter 19).
difference is important in the solubilization of The formation of phospholipid in mucosal
feed particle-bound fatty acids in ruminants, cells is predominantly by the reacylation of 1-
as the pancreatic juice contains a relatively lysophospholipids absorbed from the intesti-
low concentration of bicarbonate and intesti- nallumen.
nal pH rises more slowly in the ruminant than The newly synthesized lipids are incorpo-
in the pig. Typical pH values for the aboma- rated into lipoproteins. In the ruminant both
sum and small intestine of the sheep are 2.0 chylomicrons and VLDL are synthesized.
(abomasum); 2.5 (proximal duodenum); 3.5 Nascent lipoproteins are secreted into the lym-
(distal duodenum); 3.9 (proximal jejunum); phatic system, enter the blood stream, and are
absorbed by similar mechanisms to those against their respective concentration gradi-
described for monogastric animals. ents, and thus ATP is consumed by the mem-
brane transporter, the H+, K+-ATPase. The
chloride ions present in the stomach are
28.10 LIPID DIGESTION IN POULTRY derived from the plasma. They enter through
In poultry, lipid digestion follows a similar pat- the basal membrane against a concentration
tern to that in monogastric mammals. The main gradient via two types of membrane trans-
difference that has been observed is in the porter. The first, a bicarbonate/chloride
mechanism of delivery of absorbed lipid to the anti port, transports bicarbonate from the cyto-
blood stream. Poultry have a poorly developed plasm to the plasma in exchange for chloride.
digestive lymphatic system, and as a result the The second is a sodium/bicarbonate symport,
chylomicrons are released from the intestinal which co-transports sodium and chloride into
mucosa directly into the portal blood stream. the cell. The driving force for the accumulation
These chylomicrons are sometimes referred to of chloride ions in the cytoplasm is the com-
as portomicrons. A major consequence of this bined effect of the movement of sodium and
different absorption mechanism is that dietary bicarbonate out of the cell down their respec-
lipid passes through the liver before being cir- tive concentration gradients. The surge of
culated to the peripheral tissues. bicarbonate into the plasma during rapid HCI
secretion causes the blood plasma to become
slightly alkaline, a phenomenon known as the
28.11 DIGESTION OF PROTEINS IN 'alkaline tide'. Chloride and potassium ions
MONOGASTRIC ANIMALS move from the cytoplasm to the stomach con-
tents down a concentration gradient via a pas-
28.11.1 DIGESTION IN THE STOMACH
sive membrane symport. The high intracellu-
The stomach is the first major site of protein lar concentration of potassium ions and low
digestion in monogastric species. Two factors intracellular concentration of sodium is main-
affect the nature of proteins in the stomach: tained by the Na+, K+-ATPase. The net result
firstly the acidic environment, pH 2.0-3.0 (cor- of the concerted action of these transport
responding to [H+] concentration of approxi- mechanisms is a hydrogen ion concentration
mately 0.1-0.01 M) denatures ingested proteins; in the stomach contents which may be as
and secondly the action of the protease pepsin, much as one million times greater than that in
which partially degrades the dietary protein the plasma (pH 1.0 or [H+] = 10-1 M as opposed
into smaller peptides. to pH 7.0 or [H+] = 10-7 M).
The acid environment of the stomach is due Pepsin is secreted by the chief cells of the
to the secretion of hydrochloric acid by the stomach mucosa. The enzyme is secreted in an
parietal or oxyntic cells of the stomach inactive zymogen form, pepsinogen, and is
mucosa. This is achieved by the mechanism converted to active pepsin by the action of
shown in Figure 28.7. Carbon dioxide pepsin already present in the stomach. The
absorbed from the blood stream and produced activation involves the removal of 42 amino
in the cell by oxidative metabolism combines acid residues from the N-terminal end of the
with water to form carbonic acid. At physio- zymogen polypeptide. The secretion of prote-
logical pH (pH 7.4) carbonic acid dissociates to olytic enzymes as inactive zymogens is a pro-
produce hydrogen and bicarbonate ions. The tective mechanism which prevents proteolytic
hydrogen ions are transported across the api- attack within the secretory cells. Pepsin has a
cal membrane of the parietal cells in exchange pH optimum of around 2.0 and exhibits a
for potassium ions present in the stomach con- specificity for peptide bonds on the carboxyl
tents. Both of these ions are transported side of the aromatic amino acids phenylala-
Digestion of proteins in monogastric animals 407
HCO;
ADP+Pi
cr
cr
Figure 28.7 Transport mechanisms involved in the secretion of Hel by parietal cells of the stomach.
nine, tryptophan and tyrosine. The end prod- released in response to the presence of fatty
uct of pepsin digestion of proteins is a mixture acids and other lipids in the small intestine.
of shorter-chain polypeptides with predomi- They also inhibit acid and pepsinogen release.
nantly N-terminal aromatic acids.
The secretion of both hydrochloric acid and
28.11.2 DIGESTION IN THE SMALL INTESTINE
pepsinogen is regulated hormonally. Gastrin
production by the gastric epithelium is stimu- Partially degraded protein leaves the stomach
lated by the presence of protein in the stom- and enters the duodenum, where the digesta is
ach, by distension of the stomach, and by the mixed with pancreatic secretions that contain
autonomic nervous system. Gastrin acts upon the proteolytic zymogens, trypsinogen, chy-
the parietal and oxyntic cells to increase acid motrypsinogen, procarboxypeptidases A and B,
and enzyme release. Secretin, produced in the and proelastase. All of these zymogens are acti-
duodenum in response to increased acidity of vated by the removal of an amino acid
duodenal contents, inhibits acid and pepsino- sequence from the N-terminal end of the pro-
gen release. Two other hormones, cholecys- tein by a common activator, trypsin. This
tokinin and gastric inhibitory factor, are results in the coordinated activation of these
pancreatic enzymes and ensures that dietary to protein substrates. When this short
protein is efficiently degraded by a battery of sequence of amino acids is removed the pro-
enzymes which have differing amino acid tein undergoes a conformational change
specificities. Trypsin and chymotrypsin are which fully exposes the active site.
endopeptidases which act at various points The short peptides produced by the action
along the polypeptide chain. Trypsin cleaves of the pancreatic proteases are further
peptide bonds formed by lysine or arginine degraded in the small intestine by aminopep-
residues; chymotrypsin has a preference for tidases produced by the intestinal mucosa.
peptide bonds on the carboxyl side of pheny- These enzymes, which act both intracellularly
lalanine, tryptophan and tyrosine. The car- and in the intestinal lumen, catalyse the
boxypeptidases and elastase are exopeptidases sequential removal of amino acids from the
which remove amino acids from the ends of the N-terminal end of peptides to produce a mix-
polypeptide chain. Carboxypeptidases A and B ture of short pep tides and amino acids. In
remove amino acids from C- and N-terminal addition, the intestinal mucosa contains
ends of pep tides, respectively. Carboxypeptid- dipeptidases which act on dipeptides
ase A is specific for phenylalanine, tryptophan absorbed from the digestive tract.
and tyrosine, whereas carboxypeptidase B is Absorption of small peptides and amino
specific for lysine or arginine residues. Elastase acids from the intestinal lumen occurs via
exhibits specificity for the neutral amino acids membrane transport proteins (symports)
leucine, isoleucine and valine. which are specific for small groups of struc-
Pancreatic proteolytic enzymes are secreted turally related amino acids and also co-trans-
as zymogens to ensure that there can be no port Na+ from the lumen of the intestine into
premature activation of the zymogens before the intestinal mucosa. Na+ is usually present in
they reach the intestinal lumen. The pancreas higher concentration in the digesta than in the
also produces an inhibitory protein, pancreat- mucosal cell, and therefore moves into these
ic trypsin inhibitor, which binds very tightly to cells via passive transport, dragging the amino
the active site of any enzyme activated prior to acids with it. Once in the mucosal cell, Na+ is
release from the exocrine cells. actively transported into the extracellular fluid
The sequence of activation of the pancreat- by the action of the Na+, K+-ATPase in the
ic zymogens in the intestinal lumen is initiated basolateral membrane of the cell. This main-
by a specialized proteolytic enzyme, tains a concentration gradient between the
enteropeptidase (previously called enteroki- mucosal cell cytoplasm and the intestinal
nase), produced by the intestinal mucosa. This lumen. This process concentrates amino acids
enzyme removes a six-amino acid sequence in the cytoplasm of mucosal cells, and allows
from the N-terminal end of trypsinogen, them to move across the basal membrane and
thereby converting it to active trypsin. Only a into the portal blood stream by passive trans-
small amount of trypsinogen need be activat- port down their concentration gradient. The
ed in this way as trypsin itself will then catal- intracellular hydrolysis of dipeptides to free
yse the activation of trypsinogen and the other amino acids by dipeptidase also contributes to
pancreatic proteolytic zymogens. This regula- the amino acid concentration gradient.
tory mechanism, the activation of enzymes by Although ATP is not directly involved in the
proteolytic cleavage, has been studied in detail action of the amino acid transporters, it
for chymotrypsinogen. In the zymogen form provides the driving force for amino acid
the protein has a sequence of 245 amino acids. absorption by creating a low intracellular Na+
A 15-amino-acid sequence at the N-terminal concentration through the action of the Na+,
end of the protein appears to block the active K+-ATPase (Figure 28.8). Absorbed amino acids
site of the enzyme, preventing it from binding are transported to the liver where they may
Digestion of protein in ruminants 409
undergo a number of metabolic fates described degraded in this way to produce the
in Chapter 14. branched-chain volatile fatty acids isobutyric
acid, isovaleric acid and 2-methylbutyric acid,
respectively.
28.12 DIGESTION OF PROTEIN IN
Ammonia may be used as a convenient
RUMINANTS
nitrogen source for microbial synthesis of
Microbial proteases degrade proteins to pep- amino acids which are thereafter incorporated
tides and amino acids in the rumen. These into microbial protein. When ammonia is pro-
amino acids are utilized in two ways: they duced in excess of microbial requirements, it is
may be directly incorporated into microbial absorbed across the rumen wall, transported to
protein; or they may be further metabolized the liver and converted to urea. A proportion
by transamination and deamination to organ- of this urea is recycled to the rumen, either
ic acids, ammonia and carbon dioxide. In this directly from the blood or via the saliva, and
way they can contribute to the volatile fatty may be used as a non-protein source of nitro-
acid pool in the rumen. The branched-chain gen for microbial amino acid synthesis. Excess
amino acids valine, leucine and isoleucine are urea is excreted in the urine. The quantity of
He dependent
amino acid transporter
J
~
@) mlno@
acid amino
acid
Amino acid (Na+' K+-ATPase )

transporter
~
~ ~~
Figure 28.8 Mechanism of amino acid absorption from the small intestine.
ammonia produced in the rumen and of urea Further digestion of undegraded dietary
synthesized in the liver is dependent on the and microbial protein in the abomasum and
quantity and quality of dietary protein. Large small intestine is similar to that in monogastric
quantities of highly degradable protein lead to animals. Abomasal and pancreatic secretions
the rapid production of ammonia in excess of are similar to those in non-ruminants; how-
the capacity of the rumen microorganisms to ever, the pattern of digesta flow dictates that
use it. Under these circumstances urea excre- these secretions are produced in a continuous
tion increases. When poor quality, low-protein manner in contrast to the more intermittent
diets are fed, rumen ammonia production is secretions of monogastric species. A summary
low and the quantity of urea nitrogen recycled of protein metabolism in the rumen is shown
to the rumen by direct transfer from the blood in Figure 28.9.
and via saliva may be greater than the ammo-
nia nitrogen absorbed from the rumen. The
28.13 INHIBITORS OF DIGESTIVE ENZYMES
additional urea nitrogen is derived from the
catabolism of amino acids by the liver. Some plants contain proteins which inhibit the
The degradability of dietary proteins in the action of digestive enzymes and which may
rumen varies considerably. Some, such as result in poor animal performance when pre-
casein, are efficiently degraded in the rumen, sent in the diet. Legume seeds, for example,
whereas others such as albumin or the com- contain proteins known as protease inhibitors
plex mixture of proteins in fish meal are poor- which can inhibit trypsin and chymotrypsin.
ly degraded. The degradability of a protein is Their presence results in depression of growth
determined by a combination of factors such when raw or unheated legume products are
as physical structure, solubility characteristics, fed to non-ruminant animals. The inhibitors
and amino acid composition and sequence in are inactivated in the rumen, so their presence
relation to the substrate specificity of the in ruminant feeds does not cause the same
microbial proteases. problems. Some bean seeds contain inhibitors
Dietary crude protein
Some excreted in
urine, some recycled
to rumen in saliva r - - - - . J
i
Urea Non-protein Dietary
nitrogen true protein
I
L
Degradable
l
Undegradable
true protein true protein
!
Amino acids
'----+--- Ammonia
I~
__ ~~Microbial
protein
Protein supply to
small intestine
Figure 28.9 Summary of crude protein metabolism in the rumen.
Inhibitors of digestive enzymes 411
of amylases which may impair starch diges- Their occurrence is not limited to seeds, and
tion. Because these inhibitors are proteins they protease inhibitors may also be present in
are inactivated by heating, and it is common fruits, leaves and tubers. In some cases they
practice to subject feeds containing bean prod- appear to be produced in response to damage
ucts to heat treatment prior to feeding. caused by insects.
Inhibitors such as these often act as deter-
rents to insects feeding on them the plants.
MAINTENANCE 29
29.2 Body temperature and heat production 413
29.3 Biochemical production of heat 414
29.3.1 Background heat production from the 415
maintenance of ion gradients in cells
29.3.2 Background heat production from 416
protein turnover
29.3.3 Background heat production from 418
other metabolic events
29.3.4 Heat production from muscular activity 418
29.3.5 Heat production from uncoupled 419
phosphorylation
29.1 INTRODUCTION this cost has to be met before any production

can be seen, because only when nutritional lev-
In the short term, if an animal is not fed it starts
els are above maintenance will there be any
to lose weight as body tissues are utilized to
accretion of tissues. The maintenance level of
provide for its metabolic needs. In the longer
nutrition is usually expressed in terms of ener-
term it will die. On the other hand, if an animal
gy, and is the energy requirement of a non-
is very well fed its body weight will increase by
productive animal (i.e. one that is not growing,
the processes of growth. At a dietary level in
producing milk or laying eggs, etc.) at the point
between these two extremes there is a point at
where the dietary energy intake exactly equals
which the animal's body weight remains con-
stant. This is known as the maintenance level energy output in various forms:
of feeding. (Some authors define maintenance • heat production
as the point at which the body's energy con- • losses in faeces
tent is constant; similarly, it can be defined as • losses in urine
the point of constant body protein composi- • losses in hair and skin.
tion. The agricultural definition of constant
body weight provides a compromise which is Of these losses the largest are the first two.
very useful in farming practice.) Even though The losses in faeces will depend largely upon
body weight may be constant, the animal will the nature of the animal's diet, although there
in fact change its body composition, usually is some contribution to the faeces that comes
losing fat and adding water and protein. from the animal's body by the passage of
In agriculture there are circumstances when sloughed cells of the gut.
a maintenance level of feeding may be desir-
able, for instance breeding males may need to
29.2 BODY TEMPERATURE AND HEAT
be kept at a near-constant weight over a period
PRODUCTION
of many months or even years. Maintenance
represents the cost, in nutritional terms, of All of the animals commonly used in agricul-
keeping the animal alive. In growing animals, ture are homoeothermic - they maintain their
414 Maintenance
body temperatures within precisely defined the demands of sweating will further increase
limits. The actual mean temperatures recorded the heat load on the animal. The figures for the
vary from species to species: man (37 C) has a
0
upper and lower values will vary depending
slightly lower body temperature than most on a number of factors including species,
livestock such as cows and sheep (38.5 and breed, acclimatization, level of production, etc.
39S C, respectively) and much below the hen For example, for a temperate dairy cow the
(41 C). Temperatures can change with physi-
0 lower temperature may be as low as _150 C,
ological state and in response to the environ- whereas for a tropical breed it may be + 10 C.
0
ment. This is very clearly seen in the camel, The upper critical temperature for a temperate
which can vary its temperature over a range of dairy cow is about 20 C and for a tropical ani-
0
about 50 C during the daily fluctuation in mal it may be in the high twenties.
ambient temperature. In most animal habitats Like many other metabolic processes, heat
and under most agricultural systems, the ani- prod uction is proportional to the animal's
mal's body temperature is below that of the metabolic body weight, as can be seen from
environment. Even most tropical climates the interspecies comparisons in Figure 29.2.
have a mean ambient temperature below body The slope of the line is approximately 0.67,
temperature. Heat production is therefore a which is slightly different from the power of
very important function of the body and one 0.75 often used for calculations of metabolic
which has to rely upon biochemical reactions. body weight.
In contrast, there are situations in which the
heat produced by an animal under hot condi-
29.3 BIOCHEMICAL PRODUCTION OF HEAT
tions can exceed the animal's ability to lose
heat to the environment. The animal must Heat production can be divided into two
invoke extra cooling mechanisms that are areas:
themselves dependent upon the expenditure
• heat produced as a by-product of metabo-
of biochemical energy.
lism;
The heat produced by an animal which has
• heat produced in response to a perceived
no dietary input is known as its fasting heat
need of the body to raise its temperature.
production. A beef bull of 650 kg body weight
produces about 41 MJ daTI - roughly the Under many conditions the heat produced
same output of heat as a single bar (500 W) as a by-product of metabolism will be sufficient
electric heater running continually. Most of to meet or exceed the needs of the animal for
this energy comes from the hydrolysis of ATP heat. Where the animal is at or above the upper
(molecular weight = 414) which produces a critical point, energy has to be expended to dis-
free energy change of about 30 kJ for every perse the excess heat. In an animal that is kept
mole. To produce 41 MJ the bull has to hydrol- in an environment below its critical tempera-
yse about 1344 moles of ATP, equivalent to 556 ture, extra heat is required to maintain body
kg of ATP - nearly its own body weight. temperature. This requires some system that
Clearly, this can only happen if the ATP is con- can sense body temperature and another that
tinually being synthesized and hydrolysed. can communicate to the tissues that they
Figure 29.1 gives a generalized impression should produce more heat. The measuring sys-
of the heat production by an animal at various tem appears to be located in the hypothalamus,
environmental temperatures. The two impor- from which 'messages' are sent via the sympa-
tant figures for any animal are its lower and thetic nervous system and probably through
upper critical temperatures. If the environmen- hormones such as those of the thyroid. The
tal temperature drops below the lower critical detailed control mechanisms involve the inte-
temperature, then heat production will rise. gration of the efforts of many different physio-
Similarly, above the upper critical temperature logical systems, and are beyond the scope of
Biochemical production of heat 415
Ii
Deep body temperature
------------------------------------~
,/
1/1
1/1 Lower Upper
.2 critical critical
...o temperature temperature
c
o
:;::
U
:::I
'C
-'"
~
C.
-
!
GI
.s;;;
o
Heat loss by evaporation
a:'"
':rhermoneutral
. zone:.
Environmental temperature
Figure 29.1 Heat production of cattle at different environmental temperatures: heat production rises
when animals are kept in environments either hotter or colder than their thermoneutral zone. (After
Blaxter, K.L. (1989) Metabolic effects of the physical environment, in Energy Metabolism in Animals and
Man, Cambridge University Press, Cambridge, UK.)
this book (for further information see have to be actively created and continually
Ruckebusch et al., 1991. Physiology of Small and maintained by pumping sodium ions out of
Large Mammals. Marcel Dekker, Philadelphia). and potassium ions into the cell. This requires
energy in the form of ATP. For example, the
Na+,K+ ATPase found in cell membranes uses
29.3.1 BACKGROUND HEAT PRODUCTION
the energy released from the hydrolysis of one
FROM THE MAINTENANCE OF ION
molecule of ATP to transfer three Na+ ions out
GRADIENTS IN CELLS
of the cell in exchange for two K+ ions (Figure
22.5). The requirement for energy is particular-
Na+,K+- ATPase pumps
ly high in nerve cells where the transmission
Within animal cells the sodium ion concentra- of an impulse leads to the loss of the mem-
tion is held at a lower value than that of the brane potential. Estimates vary as to the pre-
surrounding fluid, whereas the potassium ion cise overall importance of sodium pumps in
concentration is much higher (Table 8.3). The basal heat production; figures of 5-50% of total
ionic gradient leads to the development of an resting body heat production have been quot-
electrical charge such that the contents of the ed by different researchers.
cell are at a negative potential in relation to
their surroundings. This charge is known as
Other ATP-dependent ion pumps
the membrane potential; in a resting nerve
cell, for instance, it is about 60 m V. Gradients It is paradoxical that exposure of animals to
of this type do not just happen by chance, they high temperatures increases their metabolic
416 Maintenance
Heat production (MJ/day)
100
.
Beef steer,. .. ' ~-Dalry cow
.'
10
Dog
. Pig.···
.'
. 'Human
... Horse
1
...
Rabbi~ ~Goose
. ·'Chicken
.. ' ··Rat
0.1
.-Mouse
0.01~-----------------------------------------
0.01 0.1 1 10 100 1,000
Body weight (kg)
Figure 29.2 Heat production in a range of mammalian species. Heat production is logarithmically related
to body size.
needs and consequently increases their heat 29.3.2 BACKGROUND HEAT PRODUCTION
production. The reason for this is to be found FROM PROTEIN TURNOVER
in the large amount of water that animals
Protein synthesis
need to evaporate in order to keep their body
temperature within the physiological limits. A growing heifer synthesizes about 1.5 kg of
In hot climates, a mature sheep can evaporate protein per day; of this the majority is broken
over 5 litres of water per day in the form of down again into amino acids, and at most only
sweat and of fluid from the surfaces of the about 0.2 kg or so may be added to the ani-
lungs. mal's body (assuming that she is growing at 1
The maintenance of ionic balance is only kg day-I). A mature cow at maintenance (not
one function of membrane pumps; other lactating or growing and not pregnant) has a
metabolites such as glucose have to be protein synthesis rate in the region of 2.0 kg
pumped through membranes in exchange for day-I. By far the largest part (about 40%) takes
Na+. These pumps too are dependent on the place in the gut, and of this about 40% is lost to
hydrolysis of ATP and consume large amounts the animal in the faeces. Muscle, skin and
of energy. other tissues account for approximately equal
Muscle is maintained in its resting state by shares of the remainder, with a smaller
the concentration of Ca2 + ions in vesicles with- amount (about 7%) being made and broken
in the sarcoplasmic reticulum (see Chapter 32). down in the liver.
This requires the continual operation of a Ca2 + A very rapidly growing beef animal may
ATPase. exceptionally gain 2.0 kg per day of body
weight but a more normal figure would be 1.0 Table 29.1 Effect of different N-
kg. Of this, only about 20% will be protein terminal amino acids on the half-
which indicates that, at the most, a rapidly life of proteins in yeast cells
growing animal will only add 200-400 g pro- Amino acid Half-life of protein
tein per day, figures which are very small in
comparison to the overall protein turnover Arginine 2 min
rates of the animal. In the whole animal, pro- Asparagine 3 min
tein synthesis accounts for between a sixth Aspartate 3 min
Histidine 3 min
and a quarter of total energy expenditure.
Leucine 3 min
Calculations using whole animals have sug- Lysine 3 min
gested that there is an energy requirement of Phenylalanine 3 min
about 7.5 kJ for every gram of protein synthe- Tryptophan 3 min
sized. This suggests that every amino acid Glutamine 10 min
incorporated requires the expenditure of Tyrosine 10 min
Glutamate 30 min
about 7 moles of ATP, which is greater than is Isoleucine 30 min
accounted for in the pathway of protein syn- Alanine >20h
thesis (see Chapter 21). This extra energy must Cysteine >20h
either be used in steps ancillary to protein syn- Glycine >20h
thesis itself, such as interchanges between Methionine >20h
non-essential amino acids, or it is due to the Proline >20h
Serine >20h
energy needed for protein breakdown. Threonine >20h
Valine >20h
Protein breakdown
From: Varshavsky, A. (1992) The N-end
rule. Cell, 69, 725-735.
Protein breakdown in cells takes place by one
of two mechanisms. One uses the range of
proteolytic enzymes located in the lysosome; It can be seen that there is an enormous dif-
the other is the ubiquitin pathway which is ference in the life span of proteins depending
ATP-dependent. Ubiquitin is itself a small pro- upon the identity of one single amino acid
tein which can attach to lysine residues in a (Table 29.1). Many of the proteins that are
protein that is earmarked for destruction. This marked for rapid destruction are enzymes that
protein-ubiquitin complex is then degraded to have a regulatory function controlled by mes-
small peptides and free amino acids by large sengers outside the cell. The rate of synthesis
multi-enzyme complexes called proteasomes. of the enzyme is modified by the external
The large amounts of synthesis and break- stimulus such as a steroid hormone, and so
down at first appear to be rather pointless in long as the enzyme is rapidly destroyed its
terms of the overall metabolic economy of the activity at anyone time is determined by the
animal, and the question has to be asked, strength of the stimulus.
why?". There are several reasons. Some pro- Some of the amino acids that are produced
teins are actually made with a deliberately by the breakdown of tissue proteins are re-
short life span, others are designed to last used for synthesis. Others are broken down to
much longer. The life expectancy of a protein urea, in which form some nitrogen passes to
molecule is marked in its N-terminal amino the urine whilst other nitrogen is recycled
acid. Figure 29.3 shows the longevity in yeast through blood to the salivary glands. The urea
cells of proteins with different N-terminal in saliva then provides a source of simple
residues. nitrogen to the microbes of the rumen.
418 Maintenance
Haif ilie of protein (m'ins)
10,000 ,--- - - - - - -- - - -- - - - - - - - - ,
Arg Asp His Leu Lys Tyr lie Ala Cys Gly Met
Amino acid at N terminal end of protein chain
Figure 29.3 The effect of N-terminal amino acids on the half-life of polypeptides in yeast. Polypeptides
with N-terminal amino acids to the left of isoleucine (He) have half-lives of 0-60 minutes, whereas those
to the right of isoleucine have half-lives of many hours.
The overall loss of nitrogen at maintenance changes are not necessarily favourable. Some
is the sum of nitrogen lost in the urine and fae- energy from these reactions will be lost as
ces. The daily extent of this total loss of nitro- heat.
gen in ruminants at maintenance is estimated Another possible source of heat comes from
at 350 mg per kgD,75. (The metabolic rate of ani- the so-called futile cycles. Some of these have
mals correlates well with their body weight already been described in relation to gluconeo-
raised to the power of 0.75 (VV0 75). On this scale genesis (Chapter 18); another, larger cycle can
the metabolic rates of mice and elephants are be put together by looking at the synthesis and
quite similar. The same exponent can be used hydrolysis of triacylglycerols, and a single turn
to compare the metabolic activity of animals of of this cycle leads to the hydrolysis of seven
the same species but of differing body weight.) molecules of ATP (Figure 29.4). From a meta-
bolic point of view, these reactions must be
29.3.3 BACKGROUND HEAT PRODUCTION carefully controlled to ensure that they do not
FROM OTHER METABOLIC EVENTS waste ATP by hydrolysing it back to ADP as
soon as it is formed. However, they also pro-
The activities of the sodium pump and of provide a method of producing heat within cells.
tein turnover are the two largest single con-
tributors to heat production, but even at their
29.3.4 HEAT PRODUCTION FROM MUSCULAR
highest they can only account for about a half
ACTIVITY
of the maintenance heat production. The rest
must come from a myriad of smaller contribu- Movement requires physical energy and in
tions. There are small, negative, free energy turn this must be supplied from chemical
changes associated with many biochemical energy by the hydrolysis of ATP. Not all of the
reactions; much of the energy that this repre- energy obtained from ATP hydrolysis is con-
sents will be coupled to other reactions so that verted to perform physical work - this excess
they can progress even when the free energy energy is lost as heat. It is tempting to think of
this heat as being wasted, but in many situa- mones noradrenaline and adrenaline by
tions the principal role of muscle may be its increasing their oxygen consumption and
ability to produce heat rather than physical hence their heat production. The effect on the
effort. muscle is an indirect one: it cannot be demon-
strated with excised muscles in an isolated sys-
tem. It has been calculated that the heat pro-
Shivering
duction from resting muscles in rats could in
It is a common experience that the human this way provide a large proportion of the
response to cold is to shiver, a behaviour pat- thermal energy output of the animal. The hor-
tern that we share with the rest of the mam- mones cause increases in the blood flow
mals. This activity leads to the production of through muscles, but their effects on oxygen
large amounts of heat from the hydrolysis of consumption are much greater. It is tempting
ATP, both by myosin and in the Ca2 + ATPase to suggest that a combination of the ATPase
(Chapter 32). In animals kept in environments activity of myosin and the Ca2 + ATPase within
below their lower critical temperature, this muscle form a convenient mechanism for heat
provides a rapid method for the conversion of production in animals.
the chemical potential energy of tissue stores
into thermal energy released by ATP hydroly-
29.3.5 HEAT PRODUCTION FROM UNCOUPLED
sis. The mechanical energy that is produced is
PHOSPHORYLATION
small in relation to the direct thermal output.
The young of many mammals, and the adults
of some (such as humans) have brown adi-
Possible non-coordinated muscle activity
pose tissue (BAT), this is darker in colour than
It has been shown that resting thigh muscles the normal white fat deposits. It appears to
from rats respond to administration of the hor- have a very specific function in relation to
Triglyceride
IVV\/VVVv"- coo
IVV\/VVVv"- coo
j
IVV\/VVVv"- coo
F.H,.....
~COOH
~ HO 1
~COOH
HOJ
6 ADPX 3 ATP ~COOH HO ATP

~3HSCOA
6 ATP 3 AMP~3 p~
~CO.SCoA
KADP
~CO.SCoA
~CO.SCOA
Fatty acyl CoAs
Figure 29.4 The triacylglycerol futile cycle which operates in adipose tissue. This leads to the hydrolysis
of seven molecules of ATP for each turn of the cycle.
420 Maintenance
heat production. Its dark colour is related to lation the message to the cell comes via the
an unusually high density of mitochondria. neurotransmitter, noradrenaline. The nora-
Brown adipose tissue is unique in having a drenaline itself is produced within the sympa-
protein, thermogenin or uncoupling protein thetic nervous system and binds to special
(UCP) which removes the link between mito- binding sites (termed 13 3 adrenoceptors) which
chondrial proton transfer and ATP generation are unique to BAT.
(see Chapter 12). The activity of this protein is The main substrate for BAT is, not surpris-
controlled in two ways. In the short term, the ingly, the fatty acids liberated from the triglyc-
activity of pre-existing protein can be erides stored in the tissue. The BAT therefore
switched on and off. After long-term stimulus, represents a combined heater and fuel-storage
the level of uncoupling protein in BAT unit.
increases. In both short- and long-term regu-
REGULATION AND MANIPULATION 30
OF GROWTH AND DEVELOPMENT
IN ANIMALS

30.1.1 General principles 422
30.2 Rates and patterns of growth 422
30.3 Muscle growth 424
30.3.1 Cellular growth as a component of 424
muscular growth
30.3.2 Protein accretion as a component of 425
growth
30.3.3 Control of protein synthesis 425
30.4 Growth of collagen 425
30.5 Growth of bone 427
30.5.1 Calcification of bone 428
30.6 Growth of adipose tissue 429
30.6.1 Growth of adipose tissue cell number 429
30.6.2 Deposition of fat within adipocytes 430
30.7 Manipulation of growth 431
30.7.1 Growth hormone and insulin-like 431
growth factors
30.7.2 Oestrogens and androgens 433
30.7.3 The J3-agonists 435
30.7.4 Glucocorticoids 439
30.7.5 Thyroid hormones 439
30.7.6 Antibiotics 440
30.1 INTRODUCTION need for teats, but they will become central to
the existence of a dairy cow in adult life. For
Growth is one of the fundamental processes this reason, most of the development of the
by which cheap agricultural inputs are con- teats does not take place until adult life. In
verted into expensive products, generally general, animals have waves of growth for dif-
meat. It is associated very closely with another ferent tissues, with a hierarchy of growth of
process, that of development. A mature animal brain, bone, muscle and fat. This hides some
is not just an expanded version of a new-born gross approximations: some bones have to
one: the various tissues of the body change in develop much earlier than others, for example,
their importance during development. To take as the brain is developing the skull has to grow
a simple example, a new-born calf has very lit- to accommodate it. Looking at the whole ani-
tle need for teats, a male will never develop a mal there are also waves of growth that
422 Regulation and manipulation of growth and development in animals
change its proportions during development. mal's age, health and genetic composition.
In general, growth moves from head to tail - External factors include climate, season, the
the front limbs and their associated tissues availability of food and water, and the necessi-
mature earlier than the rear ones. In agricul- ty or otherwise of having to perform strenu-
tural terms, this has implications for the plan- ous physical work.
ning of production, because the expensive The rates of growth are often controlled by
cuts of meat tend to be those produced at the the producer in order to ensure that the ani-
rear of the animal. mal's productivity is well adapted to the farm-
The rate of growth of an animal can be ing system. For instance, broiler chicks are
changed by dietary manipulation, altering the encouraged to grow at a very fast rate so as to
availability of the raw materials needed for achieve marketable weight in about 6 weeks.
body tissues; however the effect on the pat- On the other hand, chicks that are destined to
tern of development is much smaller. No mat- become egg layers are fed so that they do not
ter how well an animal is fed, its tissues will reach mature weight until they are ready to
tend to grow in the same order: brain, bones, start laying at about 22 weeks old. Similarly,
muscle and fat. dairy heifers are fed so as to gain a little over
0.5 kg per day, which means that they will be
at the right weight for breeding at around 15
30.1.1 GENERAL PRINCIPLES
months of age. If they are fed too well and
Tissue accretion is the process by which new reach the appropriate weight much before
tissue is laid down, and which defines the rate this, the mammary gland will not be suffi-
at which the animal grows. The rate of accre- ciently well developed to support high-yield-
tion can be thought of in terms of the overall ing lactations. By contrast, intensively fed beef
growth rate of tissue, or as the rate for individ- animals which are produced for their meat
ual chemical components such as protein, may grow at much faster rates.
lipid or minerals. Accretion is always calculat- Metabolic body weight (WO· 75) can be used
ed as the difference between synthesis and to study the relative potential of an animal for
breakdown. Even the largest accretion rate is growth. Figure 30.1 shows that as an animal
very small in relation to overall turnover. This grows, its rate of growth relative to metabolic
suggests ways in which animal growth can be body weight declines. The rate of deposition
made more efficient: either a small increase in of protein also decreases, following closely the
the rate of synthesis or a small decrease in the pattern of the rate of change in body weight.
rate of degradation will lead to a larger However, the relative rate of fat deposition
increase in growth rate. stays almost constant in proportion to meta-
bolic weight. Thus a heavier animal lays down
fat at a much faster rate than a light one.
30.2 RATES AND PATTERNS OF GROWTH
The composition of the animal's body
The term, 'rate of growth' is usually employed changes during development. To achieve this
describe the speed at which animals increase change, differences in the composition of what
their overall live weight, although it may at is added to the body during growth (known as
times be more important to think in terms of the composition of gain) must be even more
the speed at which the carcass of the animal extreme.
grows. (The carcass is the body minus the gas- Figure 30.2 shows the compositions of each
tro-intestinal tract, head, feet, skin, blood, etc.) extra kilogram of gain in a growing steer from
Rate of growth depends upon many factors, birth at about 50 kg to slaughter at about 500
both internal and external to the animal. The kg. The increase in fat and decrease in water
most important internal factors are the ani- during growth are demonstrated very clearly.
Rates and patterns of growth 423
70 14
60 12
s:..,
:xJ
jci -CD
e~ 50 10 ~ ~
DID;
~ a~
'iii .... 40 8 !!. a.
'Os:. :::J III
_.2'
o CD Fat
____________________________ 6 !!I ~
~
a.~
....CD
l!'O
CD 0
>.Q
:.. 30
ai
~:r
;: c 20 4 ~ ~
11-
'i
a::
ei
ICIIII
10 Protein 2 ~
o 0
50 100 150 200 250 300 350 400 450 500
Body weight (kg)
Figure 30.1 The relationship between body weight, relative rate of growth of body weight, protein and
fat expressed on a metabolic body weight basis (g kg-D75) in cattle,
1,000
c 800
'iii
aI
....
s:.
aI 600 DWater
Gi
~
ITD Minerals
-
CD
~
400 ~Fat
0
aI
I L:Zl Protein
--
..lie I
aI
200
0
50 100 150 200 300 400 500
Live weight (kg)
Figure 30.2 Composition of weight gain in a growing steer from 50-500 kg live weight.
424 Regulation and manipulation oj growth and development in animals
The rate of increase in the amount of protein adult, the nuclei remain relatively constant but
depends very much upon the quantity of the amount of protein per nucleus increases.
dietary amino acids available to the animal.
The question then arises as to what the
30.3.1 CELLULAR GROWTH AS A COMPONENT
animal does with quantities of nutrients sup-
OF MUSCULAR GROWTH
plied in the diet which exceed its ability to
deposit protein. Amino acids that are sup- In addition to the contractile cells, muscle con-
plied in excessive amounts are deaminated tains other cells such as those that surround
(Chapter 14) and the resulting a-ketoacids are the blood capillaries, fat cells, and a special
used as substrates for energy-yielding type known as satellite cells. Muscle fibre cells
processes. This means that an excessive sup- are multinuclear, so their growth cannot take
ply of amino acids becomes an over-supply of place by the simple processes of mitotic cell
energy, for which there are two possible out- division (see Chapter 32).
lets: the animal can increase its heat produc- Muscle cells begin their existence in foetal
tion (see Chapter 29), and the substrates can life as myoblasts, cells with a single nucleus
be used as precursors for the synthesis of fat and few of the characteristics of muscle. As
which is deposited in adipose tissue. Once foetal development progresses, the myoblasts
the maximum capability for protein synthesis start to fuse, forming multinuclear cells. They
is reached, any further increases in nutrition- also begin to produce some of the proteins,
al input lead to the production of a fatter car- such as actin and myosin, troponin and
cass. This situation can arise very easily in pig tropomyosin, characteristic of functional mus-
production and has also been shown to occur cle. A collagen sheath of connective tissue
in sheep. forms around the developing cells and they
take on the characteristic form of muscle cells.
At the same time the developing muscle is
30.3 MUSCLE GROWTH
invaded by nerves. The presence of nerve
The function of muscle and the structures of fibres seems to be essential for the further
its major proteins are considered in Chapter development of the muscle, and it has been
32, but the tissue's importance in agriculture is suggested that the nerve cells secrete growth
largely as the most saleable part of meat, and factors that change the pattern of muscle
thus its growth is a primary consideration for development.
the producer. Muscle is composed mainly of In agricultural species, both mammals and
bundles of muscle cells surrounded by a colla- birds, the number of muscle fibre cells is estab-
gen sheath which is continuous with the ten- lished at birth, and as the animal grows these
dons that are attached to the skeleton. Muscle fibres increase in length and width. This
growth takes place by two processes, one growth is accompanied by an increase in the
involving an increase in the number of cells, number of nuclei per cell. Work with radioac-
and the other an increase in the amount of tively labelled compounds has shown that the
protein within each muscle cell. The DNA con- new DNA does not originate inside the muscle
tent in muscle remains relatively constant dur- fibre but is passed over from the accompany-
ing juvenile growth, indicating that muscle ing satellite cells.
protein content and numbers of cell nuclei are In younger animals satellite cells are able to
growing at about the same rate. However, in undergo mitosis, and therefore growth and
later life the level of protein increases at a far development of muscle fibre cells depends
greater rate than the number of nuclei. Thus upon the division of this different type of cell.
growth in the juvenile is a mixture of protein As an animal matures, the proliferation of
synthesis and nuclear proliferation. In the satellite cells (and therefore any increase in
Growth of collagen 425
overall muscle DNA content) comes to an end. 60S units combine to form the ribosome. The
At about the same time there is a tendency for eIF-2 protein is reactivated by phosphoryla-
animals to start to deposit lipid rather than tion under the influence of a protein kinase
muscle fibres. enzyme. This allows a further cycle of
polypeptide synthesis to be initiated. The
involvement of an allosterically modified
30.3.2 PROTEIN ACCRETION AS A
kinase provides a point at which protein syn-
COMPONENT OF GROWTH
thesis becomes susceptible to control by a 'sec-
Muscle proteins are continually formed only ond messenger' system.
to be broken down again as part of a large- There are other possible control points in
scale turnover of body resources. The net the regulation of translation which may
deposition of protein is determined by the involve other initiation factors, although the
rates of protein synthesis and protein degra- evidence is, as yet, rather uncertain.
dation. When considering how muscle protein
is laid down, the ways in which synthesis and
30.4 GROWTH OF COLLAGEN
degradation are controlled must be considered
separately. The overall process of protein syn- The synthesis of collagen is central to the
thesis is described in Chapter 21, and only the growth of almost all animal tissues because it
potential points of control are considered accounts for over a quarter of the protein in
below. The degradation of protein is consid- the body. Much of the collagen is in tissues
ered in Chapters 29 and 32. such as skin and bone that have a low com-
mercial value. Despite this, such tissues are
important to the overall chemical economy of
30.3.3 CONTROL OF PROTEIN SYNTHESIS
the animal because they are vital to survival.
The pathway for protein synthesis can be split Collagen also is important because its synthe-
into two events: transcription of DNA into sis diverts amino acids away from the forma-
mRNA, and the translation of the information tion of muscle fibre protein.
contained in the mRNA into the form of The term collagen relates to a family of at
polypeptides. In broad terms, controlling tran- least 14 different proteins which are present in
scription alters the types of proteins formed, various tissues. The most widespread colla-
whereas the regulation of translation changes gen, denoted as collagen I, is found in bone,
the rate at which proteins are synthesized. tendons, skin and the cornea.
Translation takes place through the ribo- The importance of collagen lies in its physi-
some cycle (Figure 30.3) and the main point of cal properties, particularly its insolubility in
control is exerted at the initiation of peptide- water and its physical strength under tension.
chain synthesis, which takes place when the These unusual properties are matched by
405 subunit of the ribosome binds to various some extraordinary features in its chemical
initiation factors (eukaryotic initiation factors, structure. The main distinguishing features
eIFs).The first amino acid incorporated into are its atypical amino acid composition and its
the nascent polypeptide is always methionine quaternary structure.
linked to a special tRNA, which differs from Collagens have a very high content of
the tRNA which is used to carry methionine glycine which constitutes about 30% of the
when it is to be incorporated into the middle amino acid residues in the molecule. It also has
of a polypeptide chain. The 405 ribosomal sub- very high concentrations of the hydroxy
unit also has to carry an active (phosphorylat- amino acids hydroxyproline and hydroxyly-
ed) version of eIF-2. The eIF-2 complex is sine. These are not incorporated into the
released in an inactive form when the 405 and polypeptide during elongation, rather they
,IF·1 ~et.IRNA
___ A DP
~
1
. / elF.3 e1F.4F- - - - - -:IF.3 eIF-4F
/f -- Met + ATP
:.: .IF", .IF" .'F.'~NA OR.. .'F.'_

\ e1F-3 J eIF-4F
elF eukaryotlc Ribosome with

Initiation Factors 405 and 60S subunits
Figure 30.3 Initiation factors involved in protein synthesis in eukaryotic cells_ elF, Eukaryotic initiation
factors.
are formed by the modification of the amino glucose and galactose) is the hydroxyl group
acids after the translation step of protein syn- of hydroxylysine.
thesis. Hydroxyproline is formed from proline Collagen is formed from a group of
under the influence of the enzyme prolyl polypeptide chains known as a-chains which
hydroxylase. The enzyme has iron at its centre intertwine to form a triple helix. This shape is
and requires molecular oxygen (02)' reminiscent of a rope, a man-made structure of
a-Ketoglutarate is also required and is decar- great tensile strength. Collagen I comprises
boxylated to succinate during the reaction. two chains of a type called al(I) and one of a2,
One interesting feature of this reaction is the each of which has in the region of 1000 amino
need for a supply of ascorbic acid (vitamin C) acid residues.
to maintain the iron in the II oxidation state. It The constituent polypeptide chains of colla-
follows that a shortage of vitamin C will lead gen do not spontaneously form the triple
to a reduction in collagen synthesis, with con- helix, and thus the association must be one
sequences for those tissues such as skin that which is guided enzymically or by some form
have high collagen contents. Many of the of chaperone protein. The process starts with
symptoms of vitamin C deficiency do indeed the formation, in the endoplasmic reticulum,
involve lesions of skin and other tissues with of a triple helix using three polypeptide chains
high collagen levels (Chapter 8). which are considerably longer than those that
Collagens are actually glycoproteins, will eventually be found in the collagen. This
although the number of sugar residues is precursor collagen is known as procollagen.
small in comparison to the quantity of amino Once formed, it is secreted from the cell into
acids. The point of attachment for the carbo- the extracellular space, where the extra amino
hydrate (mainly mono- or disaccharides of acid residues are trimmed off under the action
Growth of bone 427
of specific pro collagen peptidases. The result- materials such as glass-reinforced plastic
ing molecules are termed tropocollagen: these (fibreglass) where the mineral glass exists in a
then associate together to form the collagen polymer matrix of resin. In bone, the mineral is
fibres. The chains of collagen are held firmly mainly calcium phosphates and the polymer
together by hydrogen bonding in a very regu- matrix is collagen. Bone does not just provide
lar pattern, with individual tropocollagen mol- the rigid frame of the skeleton, it also func-
ecules displaced from one another by about a tions as a shock absorber against mechanical
quarter of their length (Figure 30.4). damage and as a reservoir for the storage of
Collagen is rarely sold as a primary agricul- minerals, particularly calcium and phospho-
tural product but it does form the raw materi- rus. The matrix of protein gives to bone the
al for a very important industrial commodity - necessary slight flexibility to allow it to absorb
gelatin. If collagen is boiled for an extended impact, and thus to flex under stress rather
period it separates into individual tropocolla- than to break. Calcium phosphate acts as the
gen fibres, and these then break into their rigid component and also the mineral store.
individual polypeptide strands. Once the Bone development therefore requires both
association of the polypeptide strands is lost, protein synthesis and mineral deposition. In
so too is the structure of the collagen, and the Chapter 29, it was shown that tissues are
polypeptides form themselves into random dynamic and are continually being synthe-
units with a great deal of inter-molecular sized and broken down - the same processes
hydrogen bonding both between polypeptide occur in bone metabolism.
chains and between the chains and water mol- The size of the carcass of an animal is large-
ecules, so as to form a continuous gel phase. ly determined by the size of its long bones
Heating the gelatin leads to a loosening of the such as those of the legs, and these will be
hydrogen bonding and the melting of the gel. treated as the examples in this chapter. The
Gelatin is the basis of a huge number of prod- mature size of an animal is fixed at or near
ucts in the food, confectionery and pharma- puberty, and at this point there are big
ceutical industries. changes in the physiology of the bone.
A simplified form of the structure of bone is
shown in Figure 30.5.
30.5 GROWTH OF BONE
Bone formation is determined by two types
The final stature of an animal is linked to the of cells, osteoblasts that synthesize bone and
size of its skeleton: if bone growth is reduced osteoclasts that break it down. The processes
an animal's potential to deposit muscle is also start in embryonic life with the formation of
reduced. Bone is not simply a mineral materi- cartilage (largely collagen) surrounded by a
al, it is much more closely related to composite primary bone collar synthesized by the
Figure 30.4 The arrangement of individual collagen chains in a collagen fibre.

articular cartilage
epiphysis
epiphyseal cartilage osalfylng
diaphysis
marrow cavity
hard bone of shaft
cancellate bone
Figure 30.5 Simplified diagram of the structure of a long bone.
osteoblasts. This solid structure is penetrated ion may be replaced by fluoride. Some phos-
by other cells, amongst them osteoclasts, that phate is replaced with carbonate (C0 32-). Pure
hollow out an internal cavity where the cells of hydroxyapatite with a Ca:P:OH ratio of 5:3:1
the bone marrow develop and grow. This would be a solid with a hexagonal crystalline
remodelling process continues towards the structure, with planes of weakness (cleavage)
ends of the bone. At each end (the epiphyses) such that it would be more liable to fracture in
an area of actively growing bone, known as one direction than another. A pure crystalline
the epiphyseal growth plate, is formed. Within structure in bone would be disadvantageous
the growth plate, cartilage-secreting cells and therefore much of the mineral that is laid
(chondrocytes) proliferate and extend towards down is as very small individual crystals,
the shaft of the bone (diaphysis). 0.02-0.03 mm in length. This lack of consoli-
dated crystal structure is seen particularly in
younger animals where the mineral compo-
30.5.1 CALCIFICATION OF BONE
nent may be almost completely amorphous.
The mineral portion of bone consists largely of As animals mature the degree of hydroxyap-
hydroxyapatite which is a complex calcium atite crystallisation increases. The first crys-
hydroxy phosphate. One commonly quoted talline mineral to be formed may not be
formula is Ca lO (OHMP0 4)6' but this is only an hydroxyapatite itself but simpler calcium
approximation. A small proportion of the cal- phosphates (tricalcium phosphate and octacal-
cium may be replaced by another of the alkali cium phosphate), with transformation to
earth metals, mainly magnesium, but under hydroxyapatite taking place subsequently.
certain circumstances strontium may be laid Bone mineralization takes place in the
down within the crystalline structure. extracellular matrix of the bone, which exists
Similarly, a small proportion of the hydroxyl as an aqueous gel phase due to the presence of
Growth of adipose tissue 429
collagen. Also present are smaller amounts of tent compared to protein, the deposition of 1
a large range of other proteins, many of which kg fat is more expensive, in terms of the sup-
are capable of temporarily binding to (seques- ply of dietary energy, than the deposition of 1
tering) calcium. The formation of the calcified kg protein.
material occurs by a process of precipitation As observed in Chapter 9, adult animals
which needs four components: tend to lay down more fat than young ones.
Thus, farm animals are often taken to slaugh-
• a source of extracellular calcium;
ter before they reach full maturity so as to
• a source of extracellular phosphate;
restrict the amount of fat in the carcass.
• a solid phase that can act as a centre of crys-
Most of the adipose tissue in the carcasses
tal nucleation;
of farm livestock is of the white variety.
• a matrix which can form the shape of the
Amounts of brown adipose tissue are small in
growing crystals.
the carcasses of all but the new-born, thus this
In growing bone, calcium and phosphate section concentrates almost entirely on the
are released into the extracellular space from white adipose tissue. This is distributed
the osteoblasts of the epiphyseal growth plate. between a number of sites which vary in
The nucleation of crystal formation is thought quantitative importance during the animal's
to be provided by a family of proteolipids that life. Intermuscular fat develops at an earlier
contain phospholipid-phosphate complexes. age than perinephric or subcutaneous adipose
Once crystal growth has been initiated, its ori- tissue.
entation and the size of the crystals appear to The growth of adipose tissue, as with most
be shaped by the collagen matrix. tissues, rests upon two events: the increase in
the number of specific adipose tissue cells (the
hyperplasia of adipocytes); and the quantity of
30.6 GROWTH OF ADIPOSE TISSUE
fat that is deposited in each cell (hypertrophy).
Adipose tissue growth is a very topical issue in There has been much debate about the relative
the study of human biochemistry, because of importance of these processes. It was previ-
the great interest in preventing the excessive ously believed that adipocyte number was a
development of lipid deposits which are con- constant determined at birth, and that the
sidered both unhealthy and unsightly. Interest growth of fat deposits was purely a process of
in the adipose tissue of livestock is of a differ- hypertrophy. This now appears to have been a
ent nature. A moderate amount of fat deposit- mistaken assumption.
ed within meat is regarded as a commercially
valuable indicator of quality. Dietary energy
30.6.1 GROWTH IN ADIPOSE TISSUE CELL
provided as fat is deposited very efficiently as
NUMBER
carcass fat with a relatively small energy cost in
biochemical terms to the animal. This is The main source of adipocytes in white adi-
because fatty acids may be absorbed and incor- pose tissue is a line of cells known as adipocyte
porated in fat depots with little modification. precursor cells (preadipocytes). These can be
In contrast, the conversion of dietary protein to extracted from the adipose tissue of a wide
tissue protein has a much higher energy cost range of animals and cultured in laboratory
because of the high ATP consumption associat- media where they will divide, doubling their
ed with protein synthesis. numbers approximately every 26 hours. After
Excessive fat deposited in the carcass is a few weeks in culture they acquire the char-
removed during butchering. This is wasteful acteristic biochemical features of adipocytes,
in terms of the utilization of dietary energy. particularly the presence of lipoprotein lipase.
Because carcass fat has a very low water con- In addition, in humans it has been shown that
mature adipocytes are capable of DNA replica- activity of which is regulated by numerous
tion, indicating the possibility of increasing hormonal factors. The l3-adrenergic receptor-
adipocyte numbers by the simpler process of mediated activation of this enzyme is
mitotic cell division. described in more detail in section 30.7.3, and
A number of locally acting hormones is shown in Figure 30.1I.
(growth factors) have been shown to be neces- Triacylglycerol lipase exists in two forms:
sary for the proliferation of adipocyte precur- the active, phosphorylated enzyme; and the
sor cells. Furthermore, their activity can be inactive, dephosphorylated enzyme. Con-ver-
stimulated by insulin and an insulin-like sion from the inactive to the active form occurs
growth factor (IGF-2). when the enzyme is phosphorylated by a pro-
tein kinase (sometime called the A-kinase) that
is activated by an increase in the concentration
30.6.2 DEPOSITION OF FAT WITHIN
of cyclic AMP (cAMP). The reverse process,
ADIPOCYTES
inactivation of the lipase, is catalysed by a
The control of fat deposition depends upon a phosphatase enzyme. Adenosine, and the hor-
balance between the rate at which triacylglyc- mones glucagon and adrenaline, are known to
erols are formed in adipocytes (lipogenesis) stimulate the release of fatty acids from adi-
and the rate at which they are broken down pose tissue in response to some defined phys-
(lipolysis). Figure 30.6 shows the principal iological state. Glucagon signals an overall
routes of fat metabolism in the adipocyte. The energy deficit in the animal. Levels of adrena-
precursors for fat synthesis, preformed fatty line in blood plasma are increased by stress
acids released from circulating lipoproteins by which brings with it a need for energy.
lipoprotein lipase, acetate and glucose, are Adenosine is thought to act as a local modifier
taken up from blood. These fatty acids, and of adipocyte activity.
those synthesized within the cell from glucose Insulin has an antilipolytic effect probably
or acetate, are incorporated into triacylglyc- caused by its activation of phosphodiesterase,
erols. The glycerol-3-phosphate required for the enzyme which breaks down cAMP
triacylglycerol synthesis is also derived from (Figure 30.6).
glucose. The breakdown of triacylglycerols to
non-esterified fatty acids and glycerol occurs
The control of lipid synthesis
by the action of lipases. Glycerol is exported
from the adipocyte because these cells lack the Lipid synthesis is regulated in several ways.
glycerol kinase which is required to convert it One of these depends on the availability of the
to glycerol-3-phosphate. Some of the fatty glucose for the synthesis of fatty acids and the
acids formed may be activated within the cell glycerol-3-phosphate needed for triacylglyc-
and reincorporated into triacylglycerols. This erol formation. These processes are primarily
is an example of a futile cycle, but it is one regulated by insulin, which stimulates the
which may be used as a source of thermal uptake of glucose by tissues. Control is also
energy within the animal (Chapter 29). The exerted via the enzymes involved in fatty acid
non-esterified fatty acids which are not rein- synthesis. This process is regulated by its rate-
corporated into triacylglycerols pass to the limiting step which is the transformation of
blood for circulation to the liver and subse- acetyl-CoA into malonyl-CoA under the influ-
quent use in other tissues. ence of acetyl-CoA carboxylase (Chapter 19).
Although the major changes in its activity are
brought about by citrate via allosteric regula-
Control of triacylglycerol breakdown
tion, the activity of acetyl-CoA carboxylase can
The initial step in the hydrolysis of triacylglyc- also be modified by phosphorylation through
erols is catalysed by triacylglycerol lipase, the protein kinases. The effect of phosphorylation
Manipulation of growth 431
Adipocyte Insulin
r T~oII
GIyI:eroI-3-P
' - - FMIy.c:yI-CaAs
/\
1== I F~dM
l.;! +ft _Adrena/in
Insulin --
--- /1_ - - ..
~
Glucagon
G"- + AceIIIIII
Blood G"- + Ac-...

~ ....
(ctIyIoi,oaOt. Md VLOl.)
Figure 30.6 Metabolism of triacylglycerols on adipose tissue, showing the main sites of action of hor-
mones on lipogenesis and lipolysis.
is to reduce the activity the enzyme, and there- anabolic oestrogens and androgens, l3-adren-
fore to decrease fatty acid synthesis. Thus the ergic agonists, and gut-active growth promot-
overall effect of phosphorylation of acetyl- ers such as the ionophore and non-ionophore
CoA carboxylase and triacylglycerol lipase is antibiotics.
an increase in the rate of lipolysis. Changes in
the phosphorylation state of these enzymes
30.7.1 GROWTH HORMONE AND INSULIN-
are regulated hormonally.
LIKE GROWTH FACTORS
The effects on growth and carcass composition

30.7 MANIPULATION OF GROWTH
of growth hormone (GH), also known as
The last 30-40 years have seen major advances somatotropin (ST), and of the insulin-like
in our understanding of the biochemical basis growth factors (IGFs), also known as the
of animal nutrition and the endocrinology of somatome dins, are interrelated and often dif-
growth at a tissue level. This has led to the ficult to distinguish. Growth hormone is a pro-
potential to manipulate animal performance tein hormone (20-22 kDa) produced in an
via nutritional means, and by the use of natu- episodic manner by the anterior pituitary. A
rally occurring and synthetic compounds such number of animal tissues have been demon-
as natural and recombinant growth hormone, strated to have GH receptors. For many years
it was believed that growth hormone was the metabolism. Studies of the whole animal clear-
primary determinant of animal growth. Until ly indicate increased protein accretion and
recently, studies on the effects of growth hor- improved utilization of feed for protein depo-
mone have been limited by the need to extract sition. However, it is still not known how
the hormone from pituitary glands; however, these gross changes are related to intracellular
the advent of recombinant DNA techniques changes in protein metabolism. Increased pro-
has made it possible to produce growth hor- tein accretion could result from an increase in
mone in much larger quantities using modi- protein synthesis, a decrease in protein degra-
fied bacteria. It is now clear that some of the dation or a combination of both effects.
effects of growth hormone are mediated by The direct effects of IGFs on fat and protein
the IGFs, small proteins with a molecular metabolism remain largely unknown. In plas-
weight of around 7.5 kDa. Growth hormone ma, virtually all IGFs are bound to carrier pro-
acts on the liver to stimulate the production teins, the insulin-like growth factor-binding
and secretion of the IGFs, particularly IGF-l, proteins. Bound IGFs have little effect on
which then act on peripheral tissues by a clas- growth and it is the free form which interacts
sical endocrine mechanism. GH and probably with cell membrane receptors. Removal of the
other growth factors may also act on non- N-terminal amino acids from IGF-l results in
hepatic tissue to stimulate the local production the production of (des 1-3)IGF-l which is a
of IGFs, which act within the tissue by local more potent stimulator of protein synthesis
control mechanisms. In addition, GH may than the original IGF-l. This has been attrib-
have direct effects on a number of tissues. uted to a lower affinity of the plasma-binding
Chronic administration of GH generally proteins for the modified IGF, and therefore a
increases plasma insulin concentration, which higher concentration of free (des 1-3)IGF-l
would be expected to increase glucose utiliza- available to bind with cell receptors. This sug-
tion by tissues. In adipose tissue this should gests that the N-terminal amino acids are
result in a lipogenic response, and in skeletal involved in the binding to plasma IGF-I bind-
muscle provide substrates and energy for pro- ing proteins, but not with IGF-l cell mem-
tein synthesis. However, both in vivo and in brane receptors.
vitro, GH tends to decrease lipogenesis, appar- At an intracellular level the mode of action
ently by reducing glucose uptake and its of IGF-l is poorly understood. It has been
incorporation into glycerol and fatty acids. shown that the cell membrane contains dis-
Studies in vitro of adipose tissue from sheep, tinct receptors for insulin and IGFs. The IGF
cattle and pigs suggest that, despite increases receptors have been classified as types 1 and 2,
in plasma insulin concentration, GH actually but their structures and the mechanisms by
antagonizes the effects of insulin on which they initiate changes in cellular metab-
adipocytes, possibly by decreasing the sensi- olism are unknown. They may be similar to
tivity of the tissue to insulin. GH has also been the insulin receptor which is a glycoprotein
shown to increase lipolysis in some species. with four subunits, two ex and two 13. The ex
This may be due to the decrease in insulin sen- chains are extracellular, attached to the outer
sitivity and a reduction of the inhibitory effects surface of the cell membrane where they inter-
of insulin on lipolysis. As lipid accretion in adi- act with insulin. The 13 chains are transmem-
pose tissue is determined by the balance brane proteins which contain tyrosine kinase
between lipogenesis and lipolysis (see Figure activity on the intracellular domain of the pro-
30.6), the net effect of GH is the mobilization of tein. It is suggested that binding of insulin to
fatty acids from adipose tissue triacylglycerols. the receptor activates the protein tyrosine
Much less is known at the molecular level kinase. The net effect is that an intracellular
about the effects of GH on tissue protein target protein is phosphorylated on a tyrosine
residue. This protein may act directly within animals. The oestrogens (17~-oestradiol and
the cell or may be part of a second messenger oestrone) are particularly associated with
system (Figure 30.7). intact females, although they are produced
Assuming that one or both of the IGF recep- (albeit in smaller amounts) in male testes, and
tors is similar in structure and mechanism to may be responsible for the development of
the insulin receptor, there remains the ques- some male secondary sex characteristics. The
tion of how the primary signal, binding of IGF androgens (chiefly testosterone) are thought
to its receptor, is translated into a change in of as being hormones of the intact male. It is,
cellular metabolism. Measurements have however, interesting to note that the blood
shown that IGF-l is somatogenic: it increases plasma concentrations of androgens in
glucose uptake, DNA synthesis and prolifera- females may actually exceed those of the
tion of specific types of cell, e.g. chondrocytes, oestrogens. The third group of steroids, the
fibroblasts, epithelial cells and muscle satellite progestagens, do not have large effects on
cells. At the level of protein metabolism it growth.
increases protein synthesis and decreases pro- The outward effects of the sex steroids are
tein degradation. The links between these fairly simple to demonstrate by looking at the
changes and events at the receptor have yet to appearance of farm livestock which have been
be discovered. So far no second messenger castrated or left intact. For example, intact male
mechanism has been identified. animals differ in shape, with a preponderance
of muscle growth around the shoulders when
30.7.2 OESTROGENS AND ANDROGENS compared with females or castrates. The differ-
ences are not only seen in gross anatomy; in
Natural sex steroids
general entire males grow more rapidly than
Steroid hormones have a marked effect on females or castrates and produce carcasses that,
behaviour, growth and carcass composition in at the same weight, have lower levels of fat.
insulin
Tyrosine
- - - - - kNIe
domain
________. . m=ic
Figure 30.7 Mode of action of the insulin receptor.
These differences in carcass growth and nants, increases lean tissue growth and
conformation have stimulated a great deal of decreases fat deposition in dairy and beef
research into the use of steroid hormones in cows. Measurements in heifers have indicated
manipulating growth. increased nitrogen retention and protein
deposition in response to treatment.
Responses in entire males, which already have
Synthetic androgens and oestrogens
high circulating levels of natural androgens,
A number of synthetic oestrogenic and andro- are small. In ruminants, the optimum respons-
genic compounds have also been produced es to anabolic steroid treatment have been
which elicit growth responses in animals. shown to occur when oestrogens and andro-
These include the non-steroidal oestrogens, gens are given in combination where their
diethylstilbestrol (now banned from use effects appear to be additive.
because of its carcinogenic properties), hexoe- Treatment with anabolic steroids is general-
strol and zeranol, and the steroidal androgen ly less effective in pigs and poultry. In boars,
trenbolone acetate (TBA). oestrogens increase growth rate and show lit-
Numerous studies have indicated that tle effect on, or may even increase, carcass fat.
administration of naturally occurring or syn- Androgens increase lean tissue deposition and
thetic oestrogens and androgens can increase nitrogen retention in barrows and gilts, and
growth responses and cause changes in car- have variable effects on growth rates. In broil-
cass composition in farm animals. The mech- er chickens, oestrogens have the undesirable
anisms by which these changes are mediated effect of increasing body fat content.
are complex and still not fully understood.
There are clear sex and species differences in
Mode of action
the responses to treatment with these com-
pounds, which suggests that there is no sin- Oestrogens have effects similar to growth hor-
gle mechanism of action which can explain mone; it is therefore possible that the effects of
the observed growth and carcass changes. oestrogens may be mediated indirectly via
The complex interrelationships between the increased secretion of GH. This idea is sup-
component parts of the endocrine system, ported by evidence showing that oestrogen
combined with differing responses due to administration causes an increase in the size of
sex, age and nutrition, have been difficult to the anterior pituitary and elevated plasma GH
unravel - there is still much to be learned secretion in ruminants. However, oestrogens
about the mode of action of these com- must also exert their effects via other mecha-
pounds. The structures of natural and syn- nisms, as the response to the combined admin-
thetic oestrogens and androgens are shown istration of oestrogens and growth hormone
in Figure 30.8. appears to be additive. Furthermore their indi-
vidual effects on protein accretion differ. For
example, GH increases accretion by increasing
Typical responses to treatment with
protein synthesis but seems to have no effect
oestrogens and androgens
on protein degradation, whereas oestrogens
In castrate male ruminants, exogenous oestro- have little effect on (or decrease) protein syn-
gens generally increase lean tissue deposition thesis, but reduce protein degradation to a
and decrease subcutaneous and intramuscular greater extent. In addition, the administration
fat. Similar, but smaller, responses are seen in of oestrogens to rats increases plasma GH con-
females. In the entire male, body fat is often centrations but does not produce a growth
unchanged or increased by oestrogens. TBA, response, whereas direct administration of GH
the androgen most commonly used in rumi- stimulates growth.
OH
OH
Testosterone
HO
Trenbolone acetate
Oestrone
Hexoestrol
Zeranol
Figure 30.8 The structures of natural and synthetic anabolic steroidal and non-steroidal compounds.
An alternative hypothesis is that oestrogens proteins. The steroid-receptor complex is

may mimic growth hormone effects by translocated to the nucleus where it interacts
increasing the production and/or sensitivity of with specific binding sites on the DNA called
tissues to the insulin-like growth factors (IGF-I hormone-responsive elements (HREs). As a
and IGF-II). Thus it is possible that oestrogens result of the binding to the HREs, the tran-
modify the sensitivity and number of GH scription of some genes is enhanced while for
and/or IGF receptors in liver and other tissues. others it is suppressed. In this way androgens
Androgens and oestrogens also have direct and oestrogens can influence protein metabo-
effects in cells. These hormones are carried in lism (Figure 30.9).
the blood stream bound to carrier proteins.
When they reach their target tissue they are
30.7.3 THE [3-AGONISTS
released and, because they are lipophilic, pass
through the plasma membrane. In the cyto- Adrenaline (epinephrine) and noradrenaline
plasm the steroids bind with specific receptor (norepinephrine) are examples of naturally
&eroId
hormone
,, Translation
!
ofmRNA
S
metabolic
responee +-- protein
Figure 30.9 Mode of action of steroid hormones on protein synthesis.
occurring hormones, the catecholamines. most widely used compounds are clenbuterol,
Adrenaline is produced by the adrenal medul- cimaterol and ractopamine, the structures of
la and noradrenaline is a neurotransmitter pro- which are compared with adrenaline and
duced by post-ganglionic sympathetic nerve noradrenaline in Figure 30.10.
endings. These hormones have many pro- The differing and apparently tissue-specific
found physiological and biochemical effects. effects of catecholamines are, in part, due to
For example, at a cellular level they stimulate the fact that they act via different plasma
lipolysis in adipose tissue, glycogenolysis and membrane adrenergic receptors classified as
gluconeogenesis in liver, and glycolysis in «Xl' «X2' 13 1 and 13 2 which elicit different cellular
muscle. At an organ level they increase heart responses. Analogues which produce similar
rate and blood pressure and cause dilation of or greater effects than the naturally occurring
the respiratory passages. Many analogues of catecholamines are referred to as agonists. As
the natural catecholamines have been synthe- clenbuterol, cimaterol and ractopamine act via
sized for medical and veterinary purposes. In the l3-receptors they are also known by the
general, catecholamines decrease the fat con- generic term l3-agonists.
tent in animal carcasses and may increase pro-
tein deposition, resulting in the production of
Typical effects of ~agonists in meat-
leaner carcasses. Because they appear to shift
producing animals
the balance in the utilization of nutrients from
adipose tissue to skeletal muscle, they are often In general, effects of l3-agonists on carcass
referred to a repartitioning agents. The three composition are a visible reduction of subcuta-
-{}-l
HO
H
HO
'I _ ~ I •
H;-CH.-NH,
AdrenaHne Noradrenaline
ematerol
Ractopamine
Figure 30.10 Structures of the catecholamines adrenaline and noradrenaline, and the j3-agonists clen-
buterol, cimaterol and ractopamine.
neous fat cover and of internal fat depots (e.g. tages are that these compounds are very
perinephric, mesenteric, etc.). Intramuscular potent pharmaceuticals and must be handled
fat is also reduced. Typically, reductions of with great care. Typical dose levels are 2--10 mg
25-30% in fat have been observed in beef ani- kg-I in cattle and sheep diets, 1-2 mg kg-I in pig
mals and even greater reductions are seen in diets and 0.25-0.5 mg kg-I in poultry diets. In
sheep. Muscle accretion is also increased by addition, l3-agonist treatment tends to increase
treatment, and in cattle, sheep and pigs the meat toughness. It is thought that this occurs
cross-sectional area of the longissimus dorsi, a due to an increase in muscle connective tissue
measurement routinely used to assess muscle content and the cross-linking between adja-
protein accretion in animals, is increased sig- cent collagen molecules. There is also a
nificantly. In poultry the main fat depot, the decrease in the activity of proteolytic enzymes
abdominal fat pad, is reduced in size by treat- which are involved in the post mortem transfor-
ment and the protein content of the carcass mation of muscle into meat.
increases.
Advantages of the use of l3-agonists are that
Mode of action
they can be added directly to the diet or the
water supply; their effects are largely indepen- Of all the hormones, the biochemical mode of
dent of sex; and they are relatively short-acting action of the catecholamines is probably best
and so can be used at the most advantageous understood. Receptors in the plasma mem-
time during the growth period. The disadvan- brane are coupled to signal transduction pro-
teins which are called G-proteins because they causes a conformational change in the
bind to the guanosine nucleotides GOP and enzyme, converting it to its active form which
GTP. A number of different types of G-protein catalyses the first stage in the hydrolysis of tri-
have now been identified. Those involved in acylglycerols to fatty acids and glycerol
the classical adrenergic responses in cells, (Figure 30.11).
resulting in a change in the intracellular cyclic The effects of J3-agonists on protein metab-
AMP (cAMP) concentration, are designated Gs olism in skeletal muscle are less clear but may
(s for stimulation of cAMP production) and Gj be mediated via a different type of G-protein,
(i for inhibition of cAMP production). All G- Gp • This has the same trimeric structure as Gs
proteins are subunit proteins composed of and Gj but when activated by GTP binding the
three non-identical, dissociable subunits, a, J3 0. subunit interacts with membrane-bound
and 'Y. In their inactive state these proteins phospholipase C. This enzyme acts upon a
exits in their trimeric form with GOP bound to specific phospholipid component of the plas-
the 0. subunit. The 0. subunit also contains a ma membrane, phosphatidylinositol-4,5-bis-
weak GTPase activity which is involved in the phosphate, to release inositol-1,4,5-trisphos-
deactivation of the G-protein. The binding of a phate into the cytoplasm, leaving diacylglyc-
J3-agonist to a J3-adrenergic receptor sets in erol in the membrane. Both of these products
train a cascade of events which changes intra- act as second messengers within the cell.
cellular metabolic activity. Firstly, the binding Inositol-1,4,5-trisphosphate binds with specific
of the hormone to the receptor causes a con- receptors on the endoplasmic reticulum and
formational change in the receptor. As a con- causes Ca2 + channels to open, releasing
sequence, the GOP on the G-protein 0. subunit sequestered Ca2 + into the cytoplasm. The
exchanges for GTP and the 0. subunit dissoci- released Ca2+ can act in two ways. Firstly, it
ates from the J3'Y dimer. The GTP-o. subunit is can bind to the Ca2 + binding protein, calmod-
free to move in the plane of the membrane ulin. The Ca2 +!calmodulin complex then acts
and interacts with another membrane-bound on a Ca2 +/calmodulin-dependent protein
protein. In the case of the Gs-protein this is the kinase which modifies the activity of specific
enzyme adenyl cyclase. This catalyses the con- target enzymes. Some of these are involved in
version of ATP to 3',5' cyclic AMP, one of the protein synthesis and degradation. Secondly,
second messengers within the cell. The next the released Ca2+ interacts with the mem-
stage in the cascade involves the enzyme brane-bound diacylglycerol to activate yet
cAMP-dependent protein kinase, which can another protein kinase, protein kinase C,
exist in inactive or active forms. In the inactive which in turn regulates the activity of intracel-
form it consists of four subunits, two regulato- lular enzymes, some of which may be
ry subunits and two catalytic subunits. The involved in protein turnover (Figure 30.12).
regulatory subunits of this tetrameric form In addition to these J3-adrenergic receptor-
mask the active sites on the catalytic subunits, mediated effects, J3-agonists may act in other
preventing expression of enzyme activity. The ways. For example, it is possible that they
regulatory subunits have binding sites for decrease the sensitivity of adipocytes to
cAMP, and when these are occupied the insulin which would have an antilipogenic
enzyme dissociates to release two free active effect. It is not thought that changes in the cir-
catalytic units. In the stimulation of lipolysis in culating growth hormone or IGFs are involved
adipose tissue, the target substrate for the in the mode of action of J3-agonists, however
active protein kinase is the inactive form of tri- they may change the sensitivity of cells to IGFs
acylglycerol lipase. The protein kinase-catal- via their effects on insulin receptors or specific
ysed phosphorylation of triacylglycerollipase IGF receptors.
GTP GOP ATP cAMP
~ cAMP-dependent
~- protein kinase
Figure 30.11 Activation of triacylglycerollipase via the j3-adrenergic receptor and Gs protein system.
30.7.4 GLUCOCORTICOIDS 30.7.5 THYROID HORMONES
Elevated levels of glucocorticoid hormones are It has been known for many years that the
usually associated with chronic stress although administration of thyroid hormones, or of
they may have other roles, for instance associ- iodinated compounds capable of stimulating
ated with lactation. Raised levels of these hor- thyroid hormone production, can increase
mones can induce muscle wasting. This may be the metabolic rate of farm animals. At the tis-
due to an increase in the rate of protein degra- sue level, thyroid hormones increase the rate
dation rather than a lack of synthesis. It is still of protein degradation. This would be expect-
not certain whether the glucocorticoids have a ed to decrease growth rate; however, animals
role in controlling protein turnover at the con- with hyperactive thyroid function actually
centrations found in normal animals. grow faster. It is possible that the overall
Glucocorticoids may have long-term effects on increase in metabolic rate actually increases
the mobilization of adipose tissue reserves by the availability of substrates for protein syn-
increasing the number of l3-adrenergic recep- thesis. This stimulation of synthesis may actu-
tors. These hormones are not used commer- ally be greater than the activation of degrada-
cially to manipulate carcass growth. tion.
Phosphatidytinosilol-1 .4-bisphosphate
Diacylglycerol
G.-protein
GTP GOP
...
•
InsTP
Endoplasmic reticulum
Figure 30.12 The effects of i3-agonists in muscle tissue mediated via the i3-adrenergic receptor/G p protein
complex;
30.7.6 ANTIBIOTICS based principally on their mode of action.

Ionophores are used almost exclusively in
Antibiotics have been used for many years for
ruminant species and, indeed, can be fatal if
veterinary purposes in farm animals. Some,
given to equine species. Non-ionophores are
particularly those used to control bacterial
used in both ruminant and non-ruminant
infection of the digestive tract, produce a
species.
growth response and this has led to the devel-
opment of a number of compounds, now in
Ionophores
widespread use, specifically designed as gut
active growth promoters. Those commonly Cells have developed mechanisms for main-
used in the livestock industry are listed in taining a relatively constant intracellular envi-
Table 30.1. They are divided into two main ronment despite quite large changes in the
groups, ionophores and non-ionophores, chemical composition of the medium in which
Table 30.1 Commonly used ionophore and non- ture in the lipid bilayer. The hydrophilic inte-
ionophore gut active growth promoters rior of the channel has dimensions that allows
the movement of protons and potassium ions
Growth promoter Alternative names
down their electrochemical gradients. This
Ionophores dissipates the proton motive force generated
Monensin Rumensin by the membrane electron transport chain,
Lasalocid Bovatec disrupting ATP synthesis and proton-linked
Salinomycin Coxistac transport systems.
Tetranasin Valinomycin is an example of a mobile ion
Narasin Monteban
Non-ionophores
carrier. This peptide antibiotic is doughnut-
Avoparcin Avotan shaped and contains valine residues which are
Bacitracin Altacin, penitracin configured in such a way that the oxygens of
Virginiamycin Staphylomycin, eskalin their carboxyl groups are arranged octahedral-
Flavomycin Bambermycin, moenomycin ly around the centre of the molecule. This
Tylosin Tylan, tylon allows it to form a coordination complex with
potassium ions, facilitating the movement of
they grow. Considerable energy is expended potassium across the membrane.
by cells to transport inorganic ions in both The ionophores used as gut active growth
directions across the plasma membrane to promoters in farm animals are lipid-soluble
maintain the electrolyte composition of the mobile carriers, but they are not pep tides.
intracellular fluid. The ability to maintain ion Monensin, the best characterized of the
concentration gradients is usually linked to ionophore gut active growth promoters, is a
the consumption of ATP via the process of polyether ionophore. Its structure and that of
active transport (Chapter 22). another commonly used ionophore, lasalocid,
The ionophores disrupt the ion equilibrium are shown in Figure 30.13.
in cells by facilitating the uncontrolled move- These molecules are flexible and the bind-
ment of ions across the cell membrane. ing of a cation initiates the formation of a cyclic
Although they vary considerably in structure lipophilic cation-ionophore complex that can
and specificity, ionophores all possess this diffuse through the lipid bilayer. Monensin
common function, and have been defined as shows preference for the transport of sodium
'organic substances which bind to polar sub- ions and as it can pass through the membrane
stances in cell membranes and act as ion trans- only when bound to a cation or when proto-
fer agents through lipid membranes'. nated, the net movement of sodium depends
Gramicidin A and valinomycin are well on the relative concentrations of sodium and
characterized ionophores. Although they are protons inside and outside the cell. Lasalocid
not used in farm animals, they illustrate the acts in a similar way to monensin but exhibits
principal mode of action and provide exam- highest affinity for potassium, and lower but
ples of the two main types: those that form ion equal affinity for sodium and calcium.
channels in the membrane, and those that act Monensin dissipates the proton gradient
as mobile carriers in the lipid bilayer. across the cell membrane, causing a decrease in
Gramicidin A is a linear peptide of 15 amino the proton motive force and intracellular ATP
acids with hydrophobic side chains. Two mol- concentration. In addition, its effect on the sodi-
ecules of gramicidin A link together, via their um ion flux across the membrane alters the abil-
N-terminal amino acids, to form a channel in ity of microorganisms to accumulate essential
the plasma membrane. The hydrophobic side nutrients via sodium-dependent active trans-
chains of the amino acids are located on the port mechanisms. A characteristic effect of
outside of the channel and stabilize the struc- ionophores on the end products of carbohy-
CH,
CH, Monensin
H,C
Lasalocid
o
HO
H'~
HO
HO
HO
a-Avoparcin
Figure 30.13 The structure of the ionophore antibiotics monensin and lasalocid, and the non-ionophore
antibiotic a-avoparcin.
drate digestion in the rumen is an increase in been shown to be due to an adaptive change in
the relative proportion of propionic acid and a the rumen microflora in which Gram-positive
decrease in the proportion of acetate. This has bacteria, the primary producers of acetate and
butryrate, are reduced in numbers, whereas the more complex peptidoglycan structure they
Gram-negative species, which produce succi- are more susceptible to the effects of some
nate and propionate, increase in numbers. antibiotics than Gram-negative bacteria. The
Monensin also has direct effects on methane synthesis of peptidoglycan and the sites of
production in certain bacteria. This is due to the action of non-ionophore antibiotics are shown
dissipation of the proton gradient across the cell in Figure 30.14.
membrane which is an essential factor in The initial stage in peptidoglycan synthesis
methane production. The shift in the microbial is the formation of a UDP-N-acetylmu-
population from acetate producers to propi- ramylpentapeptide. The second step in the
onate producers also contributes to the synthesis involves the transfer of the N-acetyl-
decrease in rumen methane production, as pro- muramylpentapeptide from UDP to the iso-
pionate synthesis from pyruvate uses hydrogen prenoid compound undecaprenol phosphate
which would otherwise be lost as methane. and the subsequent formation of the dis-
sacharide repeating unit by the addition of N-
acetylglucosamine. The final step before trans-
Non-ionophores
port of this building block across the plasma
The non-ionophore antibiotics used as gut membrane is the sequential addition of five
active growth promoters in livestock produc- glycine residues to the pentapeptide.
tion have more complex structures than the Undecaprenol phosphate plays a crucial role
ionophores and contain sugar and/or peptide in the translocation of this highly polar build-
groups. In general, non-ionophores show ing block across the plasma membrane. On the
more marked effects on Gram-positive outer surface of the plasma membrane, the
microorganisms in the digestive tract than on peptidodisaccharide is transferred from the
the Gram-negative bacteria. undecaprenol phosphate to the reducing end
In Gram-positive organisms, the cell wall of an existing peptidoglycan chain.
consists of peptidoglycans composed of layers Avoparcin (Figure 30.13) disrupts peptido-
of linear chains of polysaccharide made up glycan synthesis at two stages. Firstly, it
from repeating disachcharide units of N- inhibits the extracellular transfer of the pepti-
acetylglucosamine and N-acetylmuramic acid. do disaccharide to the existing peptidoglycan.
The polysaccharide chains in each layer are Secondly, at higher concentrations avoparcin
linked at numerous points by a pentapeptide also inhibits the transfer of the N-acetylmu-
consisting of four amino acids, L-alanine, D- ramylpentapeptide from UDP to un de-
glutamic acid, L-Iysine and D-alanine. To caprenol phosphate. Avoparcin was banned as
strengthen the peptidoglycan structure fur- a feed additive in the countries of the
ther these short peptides are also cross-linked European Union in 1997.
by a glycine pentapeptide. When released from the peptidodisaccha-
In Gram-negative organisms the cell-wall ride the undecaprenol is in the pyrophosphate
structure is simpler, consisting of a single layer form, and must undergo dephosphorylation
of peptidoglycan sandwiched between the to the monophosphate before it can be recy-
inner plasma membrane and an outer lipid cled to the cytoplasm where it can react with
membrane containing large lipoproteins, another UDP-N-acetylmuramylpentapeptide.
which extend through the peptidoglycan layer Bacitracin inhibits this recycling process.
and are anchored in the plasma membrane. Bacitracin also inhibits other bacterial reac-
A number of antibiotics, including some of tions which use undecaprenol phosphate. It
the non-ionophore gut active growth promot- also forms a complex with the eukaryotic
ers, inhibit bacterial growth by disrupting the equivalent of undecaprenol, dolichol, which is
assembly of the peptidoglycan layer. As used in the synthesis of cell membrane glyco-
Gram-positive microorganisms contain a proteins. Much higher concentrations of baci-
tracin are required to affect eukaryotic cells to undecaprenol phosphate which interferes
compared with prokaryotic cells. with the synthesis of proteoglycans. It inhibits
Flavomycin is a phosphorus-containing gly- the glycosylation reaction which adds N-
colipid with considerable structural similarity acetylglucosamine to the undecaprenol phos-
"~
BacitracIn X
I
Avopereln
Figure 30.14 Site of action of the non-ionophore antibiotics on the synthesis of peptidoglycan.
phate-linked N-acetylmuramylpentapeptide, the digestive tract. In ruminants, the shift in
probably by substituting for undecaprenol fermentation pattern increases the availability
phosphate and forming an unreactive ana- of propionate for hepatic gluconeogenesis and
logue. The outer membranes of Gram-positive thus increases the supply of glucose to the
bacteria form an effective permeability barrier peripheral tissues. The increased supply of
to flavomycin, and this type of bacterium is less propionate and/or glucose may alter the pro-
susceptible to flavomycin action. file of tissue metabolism possibly mediated via
Virginiamycin and tylosin act via a different insulin and growth hormone. The decrease in
mechanism: they are both potent inhibitors of methane production conserves dietary ener-
protein synthesis. Virginiamycin consists of gy, effectively increasing the metabolizable
two components, virginiamycin M and vir- energy content of the diet. Ionophores and
giniamycin S. The M and S components bind non-ionophores usually reduce rumen ammo-
tightly to the 50S subunit of prokaryotic ribo- nia concentration indicating a decrease in the
somes. The M unit appears to block the P and degradation and deamination of dietary pro-
A binding sites on the ribosome to prevent tein sources. Although the total amount of
binding of the aminoacyl-tRNAs and the sub- protein degraded in the small intestine does
sequent peptidyltransferase reaction. The not increase, the composition of the protein
mode of action of the S unit is not fully under- may change due to reduced microbial protein
stood but is appears to be different from that synthesis in the rumen and the escape of
of the M unit. In addition, virginiamycin caus- dietary protein from rumen degradation. The
es the breakdown of polyribosomes (see resulting shift in the composition of amino
Chapter 21 for detailed coverage of protein acids available for absorption in the small
synthesis). Tylosin has a similar mode of intestine is considered to improve the overall
action. It binds to the 50S ribosomal subunit efficiency of protein deposition.
and is a potent inhibitor of ribosomal binding The use of antibiotics as growth promoters in
of aminoacyl-tRNA and the tRNA linked to farm animals is a subject of some controversy.
the nascent peptide. It also inhibits the pep- There is growing concern about the increased
tidyltransferase reaction. incidence of bacterial pathogens which are
The growth improvements seen in animals resistant to a broad spectrum of common
in response to the use of these antibiotics are antibiotics. Many of these microorganisms also
due mainly to changes in nutrient availability occur in animal species and may have devel-
in the gut, but the underlying cause of these oped some antibiotic resistance due to the prac-
changes is the altered microbial population in tice of adding antibiotics to animal feedstuffs.
LACTATION AND ITS MANIPULATION 31
31.2 Origins of the components of milk 448
31.3 The origin of lactose 449
31.4 Milk proteins 451
31.4.1 The origin of milk proteins 451
31.4.2 Amino acid supply to the mammary 451
gland
31.5 The fats 452
31.5.1 Synthesis de novo 452
31.5.2 Uptake of fatty acids from blood 453
31.5.3 Modifications to fatty acids in the 453
mammary gland
31.6 The supply of energy in the mammary 454
gland
31.7 Metabolism in lactation 454
31.7.1 Endocrine control of lactation 454
31.8 Manipulation of lactation 455
31.8.1 Dietary manipulation of lactation 456
31.8.2 Manipulation of milk production by 457
exogenous hormones
31.1 INTRODUCTION steadily to less than a half of maximum. In

early lactation, the cow's food intake is insuffi-
The majority of the world's supply of milk cient to meet the requirements for milk pro-
comes from cows, with water buffalo, goats, duction. Thus, the cow must contribute
sheep and camels producing much smaller metabolites from her own body reserves to
amounts. The mammary gland of a high-yield- enable milk secretion to continue. Food intake
ing animal has an extraordinary rate of meta- increases during lactation, so in the later
bolic activity, which may be greater than the months the cow is able both to lactate and to
rest of the whole of the animal's metabolism replace the lost body reserves (Figure 31.1).
put together. This metabolic activity is initiat- The peak yield may exceed 50 kg per day
ed very rapidly: the cow is able to make the which represents a daily secretion of about
transition from a non-lactating state to a high 1.65 kg of protein, 1.95 kg of fat and 2.25 kg of
milk yield within a very few days. High-yield- lactose. There is a correspondingly large
ing dairy cows are capable of producing well demand for substrates and energy to fuel the
over 10 tonnes of milk within a period of 10 synthetic processes. A comparison of these
months. During this time the rate of produc- metabolic activities with those of meat ani-
tion is not constant but builds up to a peak mals may serve to put them into context. A
over the first few months and then declines very rapidly growing beef animal of perhaps
448 Lactation and its manipulation
Milk yield or
food dry matter intake (kg/day) Body weight (kg)
25~----------------------------------~620
,
,,
, 600
20 !Body weight!
,,
",," 580
" '"
15 ,," "! Food intake!
" , ....
10
540
Food Intake not sufficient
to meet needs for lactation
514----. . 520
0~~--~--~--~--~~--~--~--~~500
o 1 2 3 4 5 6 7 8 9 10
Month of lactation
Figure 31.1 Lactation curve for a dairy cow. The milk yield increases during the first 2 months of lacta-
tion and then declines. In the early months of lactation, food intake is insufficient to meet the needs of
lactation. Body weight initially declines but recovers in late lactation.
350 kg live weight may add a maximum of 0.3 31.2 ORIGINS OF THE COMPONENTS OF
kg of protein to its body mass each day. A MILK.
sheep of 30 kg live weight may gain 50 g pro-
tein per day. Assuming that the animal's The mammary glands of animals vary greatly
metabolic activity is related to its metabolic in their outward size, shape, number and even
body weight (WO. 75), then the protein deposit- anatomical location, but their internal struc-
ed by fast-growing ruminants such as cattle or tures are very similar. The functioning gland
sheep is in the order of 3.5 g protein kg-O·75 • By consists of a series of secretory cells connected
contrast, the high-yielding dairy cow has a by fine ducts to larger ducts which communi-
body weight of about 600 kg which means cate with even larger ones until they meet the
that she is secreting 13.6 g protein kg-O· 75 - teat or nipple area. Figure 31.2 shows the
nearly four times as much. secretory cells, organised in clumps (alveoli)
There is a further advantage to protein surrounding a space (the lumen) into which
production from milk; in a meat animal a the milk is initially secreted prior to transport
large proportion of the protein is in the form through the duct system. Each alveolus is sur-
of collagen or in tissues that are not normally rounded by fine blood vessels, through which
eaten, and is thus not available to the eventu- the precursors required for the synthesis of
al consumer. In dairy production it can be milk are delivered. Also surrounding the alve-
assumed that all of the protein is available for olus is a network of contractile cells (myoep-
use. ithelial cells) which, in response to hormonal
The origin of lactose 449
signals, can squeeze the whole alveolus and Glucose enters the Golgi body freely from the
thus push the milk out of the central lumen cytoplasm via pores, but the other precursor,
and towards the ducts. UDP-galactose, has to be actively carried
The secretory cells (Figure 31.3) have a inwards. After the formation of lactose, the lib-
highly organized internal structure with a well erated UDP is hydrolysed within the Golgi
developed endoplasmic reticulum, numerous body to UMP and Pi' the UMP is actively
mitochondria, ribosomes and large Golgi bod- exported to the cytoplasm, and the Pi crosses
ies. The cell membrane facing the lumen (the the membrane through a pore. This completes
apical membrane), has numerous projections a uridine phosphate cycle.
(villi) which increase the surface area. At the The lactose produced is stored in the
opposite end of the cell the basal membrane lumen of the Golgi body ready for transport
lies in contact with the walls of capillary blood to the apical membrane and secretion into the
vessels. alveolar lumen.
In comparison with the other constituents
of milk, lactose has a relatively low molecular
31.3 THE ORIGIN OF LACTOSE
weight and is readily soluble in water.
The disaccharide lactose is almost unique to Therefore one of its effects is to raise the
milk. For the individual steps of the synthetic osmotic pressure to isotonic level. During
pathway, see the section on disaccharide syn- synthesis, the local concentrations of lactose
thesis (Chapter 18). The enzyme that catalyses must be much higher than they are in the
the final step, lactose synthase, is situated on final product, which would render the con-
the inner surface of the membrane of the Golgi tents of the secretory cell hypertonic. This
bodies (Figure 31.4). The active form of the unsatisfactory state is avoided by the fact that
enzyme consists of galactosyl transferase the high concentrations of lactose are
whose specificity is modified by the presence retained within the structure of the Golgi
of a-lactalbumin (see Figure 18.9). Lactose syn- bodies before transport to the apical mem-
thase has a requirement both for Mn2 + ion and brane in vesicles. As lactose is the principal
another divalent cation (X2+ in Figure 31.4). osmoregulator of milk, its secretion controls
Myoepithelial cells Alveofar
cells
Section
showing interior
of alveolus
Figure 31.2 A cluster of alveoli in the mammary gland of a goat. (Reproduced with permission from
Cowie, A.T. (1972) Lactation and its hormonal control, in Reproduction in Mammals (eds c.R. Austin and
R.V. Short), Cambridge University Press, Cambridge, UK.)
Figure 31.3 The ultrastructure of three alveolar cells and a myoepithelial cell. (Reproduced with permis-
sion from Cowie, AT. (1972) Lactation and its hormonal control, in Reproduction in Mammals (eds c.R.
Austin and R.V. Short), Cambridge University Press, Cambridge, UK.)
Glucose +~-....,:...~
UDP-glucoa.
Pf\--..J
GIUCOae-1-PhoaPhate--1.
UTP
ADP--4 ADP ATP
ATP...-1,
UDP ~U"P
Cytoplasm
Figure 31.4 Role of the Colgi body in the synthesis of lactose. The lactose synthase complex [glycosyl
transferase (CT) and a-lactalbumin (aLA)] and nucleotide diphosphatase (NDPase) are bound to the
inner membrane of the Colgi body.
Milk proteins 451
the amount of water secreted and hence the to globulin may function as a carrier for some
quantity of milk produced. of the vitamins.
a-lactalbumin is the specifier protein for
lactose synthesis, and thus in its absence no
31.4 MILK PROTEINS
lactose and hence no milk can be produced.
31.4.1 THE ORIGIN OF MILK PROTEINS Milk fat globules form at the endoplasmic
reticulum and migrate to the apical mem-
The proteins of milk are synthesized within brane. As they cross this membrane they
the gland from amino acids taken up from acquire a coating of cell membrane material. In
blood. Small amounts of blood proteins, such this way, membrane glycoproteins enter milk.
as immunoglobulins and serum albumin, can
be detected in milk. Excessive amounts of
these, particularly of serum albumin, are usu- 31.4.2 AMINO ACID SUPPLY TO THE
ally associated with 'leakage' through a mam- MAMMARY GLAND
mary gland that has been damaged in some Work has shown that the proportions of
way, possibly by the diseases known collec- amino acids absorbed from the blood are dif-
tively as mastitis. ferent from those found in milk proteins, so
In most species the commonest milk pro- within the gland there must be a large degree
teins are the caseins, which are distinguished of inter-conversion of amino acid. For
in two ways. Firstly, they have isoelectric instance, in goats the mammary uptake of
points in the region of 4 and precipitate arginine is many times the amount which is
under mildly acidic conditions: this property secreted in milk but more serine is passed to
forms the basis of the production of ferment- the milk proteins than can be accounted for by
ed milk products such as yoghurt and cheese. uptake from blood. Both of these are non-
The second unusual feature is that they are essential amino acids. The essential amino
highly phosphorylated. In milk, the negative acids are either supplied in amounts equal to
charges on the phosphate groups are neutral- their secretion in milk, or are oversupplied.
ized by the presence of calcium (Ca2 +) ions. Amino acids are taken up by the secretory cells
This means that, in addition to supplying the by active transport.
young growing animal with a dietary supply
of amino acids, casein also contributes much
31.4.3 PROTEIN SYNTHESIS
to its needs for calcium and phosphorus. The
caseins of cows' milk can be separated by The synthesis of the proteins of milk follows
electrophoresis into at least five bands, denot- the normal, ribosomal pattern (see Chapter 21)
ed; a S2-' a S2-' ~-, "1- and K-. The a S1 - and ~- frac- -what is unusual about the mammary gland is
tions are present in the largest amounts (see that a very high proportion of the ribosomes
Figure 31.5). are attached to the membranes of the endo-
If the pH is reduced to approximately 4 plasmic reticulum. Milk proteins are synthe-
then all of the caseins are precipitated. The sized with a signal' peptide of 15-21 amino
I
proteins that remain in solution are known as acids at the amino-terminal end of the grow-
the milk serum or whey proteins. The major ing chain. This peptide is recognised by recep-
protein of the whey fraction is ~-lactoglobulin. tors on the membrane of the endoplasmic
This has an important role in the nutrition of reticulum, allowing it to be transported
the young due to its relatively high levels of through the membrane with the simultaneous
sulphur amino acids, which help to balance removal of the signal sequence by a protease.
the low and potentially limiting levels found It is at this point that any sugars are added to
in casein. There is also a suggestion that ~-lac- form the cell membrane glycoproteins.
g/kg of total protein

SSO
cr., a.a IJ 'Y K a-LA /S-LO Other.
C.s.lns Serum or whey

protein.
Figure 31.5 Quantities of individual protein fractions in cow's milk.
Caseins have a high concentration of phos- 31.5.1 SYNTHESIS DE NOVO

phate groups which are attached to serine
Fatty acid synthesis in the cow takes place by
residues in the polypeptide chain, after its the elongation of a primer molecule of acetyl-
transfer to the Golgi body, by the activity of a CoA by the sequential addition of pairs of car-
casein kinase enzyme. K-casein is also a glyco-
bon atoms from malonyl-CoA (see Chapter
protein and probably receives its carbohydrate 19). In non-ruminants the major part of the
moiety within the Golgi body.
carbon for fatty acid synthesis is supplied by
acetyl-CoA which has been formed from glu-
31.5 THE FATS cose by glycolysis. On the other hand, rumi-
nants gain much of their nutrition in the form
Most of the fat in milk is in the form of triacyl- of the volatile fatty acids produced in the
glycerols, together with far smaller amounts of rumen, particularly acetate, propionate and
phospholipid, mono- and diacylglycerols, and butyrate. Only one of these, propionate, can
steroids. The fatty acids of milk have their ori- be used as a supplier of glucose, but the other
gins in two distinct sources: two are excellent sources of acetyl-CoA and
• synthesis of fatty acids within the mam- can easily be used as precursors for fatty acid
mary gland itself (sometimes called synthe- synthesis. Acetate is absorbed through the
sis de novo); rumen wall and circulates to tissues such as
• uptake from the blood of fatty acids that the mammary gland, where it is activated by
have been synthesized or stored in other the formation of its CoA ester.
tissues. acetate + CoASH + ATP -> acetyl-CoA +
AMP + PP j
In general, about 40% of fatty acids are syn-
thesized in the mammary gland and 60% The reaction is catalysed by acetyl-CoA syn-
transported in blood as pre-formed units. thetase.
The fats 453
Butyrate is absorbed into the rumen wall blood may be modified before they are incor-
but is there transformed to 3-hydroxybutyrate porated into the triacylglycerols of milk. In
(one of the ketone bodies) before it is passed to ruminants, the most important changes are
the blood stream for circulation to other tis- the de saturation at the Ll9 position of palmi-
sues. The activation of butyrate to butyryl-CoA tate (C16:0) and stearate (C18:0) to yield
and its transfer to the mitochondrial matrix are palmitoleate (C16:1) and oleate (C18:1),
similar to those shown in Figure 13.1. In the respectively (see Chapter 19). In the mam-
matrix, two hydrogen atoms are removed to mary glands of non-ruminants such as the rat
form a double bond and yield Ll2 trans- there is a large-scale elongation of palmitate
butenoyl-CoA. The reaction uses FAD as the to stearate, but this does not appear to oper-
acceptor of the hydrogens. An enoyl hydratase ate to any great extent in the mammary gland
adds water across the double bond to give L-3- from ruminants.
hydroxybutyryl-CoA, which loses its coen-
zyme group to liberate the 3-hydroxybutyrate.
Within the mammary gland, 3-hydroxybu- The distribution of fatty acids of milk fat
tyrate can be oxidized to yield acetoacetyl-CoA Milk fat is unusual in the very wide range of
which can be cleaved, with the addition of a fatty acids that it contains. These can be divid-
further molecule of coenzyme A, to give two ed into three main groups: short, medium and
molecules of acetyl-CoA. This acetyl-CoA and long, on the basis of chain length.
that produced from acetate are precursors for
fatty acid synthesis. • Short- and medium-chain fatty acids (C4:0-
C6:0 and C8:0 - C12:0). These acids are not
31.5.2 UPTAKE OF FATTY ACIDS FROM BLOOD present in circulating lipids and must be
synthesized in the gland. As under most cir-
Despite the fact that there is a high level of cumstances fatty acid synthesis produces
non-esterified fatty acids (NEFA) in the blood palmitate (C16:0), the premature release of
of lactating cows, these are not used as a short- and medium-chain acids from the
source of the fatty acids for milk. Rather it is fatty acid synthase demonstrates the pres-
triacylglycerols bound to proteins and circu- ence of a specialized thioesterase in mam-
lating as very fine emulsions in the form of mary tissue.
either chylomicrons or very low density • Long chain fatty acids (C14 and above). The
lipoproteins (VLDLs) that supply the pre- majority of these acids are supplied from
formed fatty acids. The triacylglycerol com- the triacylglycerols of blood.
ponents of these lipoproteins arrive at the
capillaries of the mammary gland where they Lipoprotein lipase provides the mechanism
are hydrolysed and the products, glycerol for the uptake of circulating fatty acids of both
and fatty acids, immediately taken up into dietary and endogenous (adipose tissue) ori-
the secretory cell. Once in the secretory cell, gin for use in the synthesis of milk fat. It is
the fatty acids move to the endoplasmic retic- interesting to note that in mammals the activi-
ulum where they are re-esterified to triacyl- ties of lipoprotein lipase in adipose tissue and
glycerols. mammary glands change in a reciprocal man-
ner. During lactation there is a marked
increase in the activity of this enzyme in the
31.5.3 MODIFICATIONS TO FATTY ACIDS IN
mammary gland and a decrease in adipose tis-
THE MAMMARY GLAND
sue. This change is probably coordinated by
Fatty acids which are either synthesized de changes in hormonal status to allow reparti-
novo in the mammary gland or absorbed from tioning of lipid to the mammary gland at a
time when substrates for milk triacylglycerol 31.7 METABOLISM IN LACTATION.
synthesis are required.
The start of lactation requires some enormous
changes in the metabolic activity of the dairy
31.6 THE SUPPLY OF ENERGY IN THE cow. During the early months of lactation the
MAMMARY GLAND cow is unable to eat enough food to supply all
of the nutrients that leave in her milk, result-
In looking at the synthesis of. the components
ing in a drop in body weight (Figure 31.1). This
of milk, it is tempting to think only of the path-
might be regarded as a prime feature of a dairy
ways in which the carbon skeletons of the lac-
cow as compared with a beef cow which, in
tose, fats and protein are formed and to ignore
response to a shortage of food, will tend to
the supply of energy, which is equally impor-
reduce her milk yield so as to ensure a meta-
tant. For lactose synthesis, ATP is required for
bolic balance.
the activation of galactose to form UDP-galac-
A cow has to partition nutrients between
tose. Similarly, in protein synthesis there are
the needs for her own body metabolism and
ATP requirements associated with the process-
the requirements of lactation. This process of
es of translation and transcription. The source
partition is carefully controlled in a dairy cow.
of this ATP is the TCA acid cycle and the elec-
As lactation starts the metabolism of tissues
tron transport chain. The synthesis of triacyl-
changes:
glycerols also requires energy in the form of
ATP to drive a number of reactions. • adipose tissue - all aspects of lipogenesis
There is, however, another large energy are reduced, lipolysis is stimulated;
requirement, this time for reducing power in • liver - large increase in the production of
the form of NADPH, for fatty acid synthesis. glucose and 3-hydroxybutyrate;
Fatty acid synthetase is located in the cyto- • skeletal muscle - protein degradation
plasm which is where the NADPH is needed. increased, glucose utilization reduced.
Most of the reducing power produced during All of the changes are designed to reduce
catabolism is in the form of NADH, and the the use of biochemical precursors in other tis-
greater part of this is formed during the opera- sues and to pass them to the mammary gland.
tion of the TCA acid cycle in the mitochondri- The control of these processes must come from
on. There are two ways in which the cytoplasm the activities of the animal's endocrine system.
can gain NADPH. One is to transform reducing
power in the form of mitochondrial NADH into 31.7.1 ENDOCRINE CONTROL OF LACTATION
cytoplasmic NADPH, and the other is to use a
pathway such as the pentose phosphate path- One point to note at the beginning of this dis-
way, which is capable of producing NADPH in cussion is that there are quite large differences
the cytoplasm. Both of theses routes are used in in the ways in which hormones controllacta-
the mammary gland although their relative tion in ruminants and non-ruminants. Much
importance probably differs between species. of the research on endocrine control of lacta-
The pentose phosphate pathway requires glu- tion has been performed on rats but, as lacta-
cose as its precursor, whereas mitochondrial tion in rodents is not a common economic
pathways use acetyl-CoA which can be formed activity, discussion here will be limited to
from acetate. ruminants. Hormones are involved in two
At the start of lactation there are large main areas in the process of lactation:
increases in the activity of fatty acyl synthetase • lactogenesis - the process by which a non-
which are paralleled by a rise in the activity of lactating gland is prepared for lactation and
the dehydrogenases of the pentose phosphate gains the ability to synthesize the specific
pathway. products of milk, e.g. a-lactalbumin;
Manipulation of lactation 455
• galactopoesis - the process by which the isolated mammary tissue. Thus its action must
body regulates the continuing lactation and be expressed through some other factor, prob-
passes precursors from the rest of the body ably IGF-l, which is released from many tis-
to the gland. sues (liver, muscle, bone) in response to
increases in growth hormone level. The mam-
Lactogenesis is a complex process and is mary gland in the ruminant has receptors for
beyond the scope of this book, the interested IGF-l, and infusing IGF-l into the blood of
reader is referred to specialized texts on lacta- goats leads to an increase in milk yield. The
tion. Galactopoesis, however, is mainly a local effects of IGF-l include:
process for the redirection of biochemical
resources. • stimulation of lipolysis in adipose tissue
There are two parts to the activity of any leading to an increased availability of fatty
hormonal control mechanism. The first is the acids for milk fat synthesis;
action of the endocrine tissue in releasing the • stimulation of liver gluconeogenesis (mainly
hormone into the bloodstream. Equally impor- from propionate);
tant is the responsiveness of the target tissue • stimulation of glycogen breakdown in
to the hormone in question, and both of these muscle.
factors have to be considered together. These factors all allow a greater part of the
animal's resources to be used in the mammary
gland.
Metabolic hormones
Insulin has a major role in the partitioning
Lactation is affected by the levels of many hor- of nutrients during lactation. However, in
mones, the principal ones are those normally ruminants insulin has little or no direct effect
regarded as metabolic hormones, although on the metabolism of mammary gland tissue.
reproductive hormones may also exert some During lactation in ruminants the level of
influence on milk synthesis. Insulin, glucagon, insulin in blood is suppressed, leading to a
growth hormone (somatotropin) and the reduction of its action on other tissues. Insulin
insulin-like growth factors (IGF-l and IGF-2) has a central role in promoting the uptake of
are intimately associated with the control of substrates by muscle and other peripheral tis-
milk synthesis. Other hormones such as those sues so that the decrease in insulin will lead to:
of the thyroid (triiodthyronine and thyroxin), • reduced uptake of glucose in insulin-sensi-
the adrenal cortex (cortisol and corticosterone), tive tissues and an increased availability to
and the adrenal medulla and sympathetic ner- the insulin-insensitive mammary gland;
vous system (adrenaline and noradrenaline) • decrease of lipogenesis in adipose tissues
may be needed for the continuation of lactation leaving more precursors available to the
and have effects within the general metabolic mammary gland.
milieu. Prolactin is required for lactogenesis
and might be expected to have an important
31.8 MANIPULATION OF LACTATION
role in galactopoeisis. It is needed for the main-
tenance of lactation in non-ruminants but Milk production from cows is a commercial
appears to have a much reduced role in the activity which usually has high costs (feed etc.)
ruminant. but results in a product of high value. The
Removal of growth hormone from blood margin between these two figures may be very
leads to a large decrease in milk production in small, and it is essential that the producer is
ruminants. Supplementation of GH leads to able to manipulate lactation in such a way as
large increases in milk output. This hormone to optimize the returns. Apart from changing
has no direct effect on the metabolic activity of the genetic composition of the herd and the
environmental conditions, there are two main High levels of dietary soluble carbohydrate
mechanisms for control. The first of these is to also lead to the formation of a rumen microflo-
modify the diet and the second is to adjust the ra with a lowered ability to hydrolyse cellu-
metabolism of the cow by the use of exoge- lose. The rate of degradation in the rumen is
nous hormones. reduced, as is the intake of food.
Accompanying these changes are reductions
in rumen pH that may lead to the develop-
31.8.1 DIETARY MANIPULATION OF
ment of acidosis. Addition of extra buffering
LACTATION
capacity to the rumen has been shown to alle-
The quantity of milk produced by the cow is viate some of the deleterious effects of feeding
determined largely by the amount and quality high-concentrate diets. A common technique
of nutrients ingested in the diet. In early lacta- is to add 1-2% of sodium bicarbonate to dairy
tion the cow uses some of her body reserves as rations, particularly in conditions where heat
a source of nutrients, particularly amino adds stress is also a potential problem.
and lipids, but the extent of this contribution is Added ionophores (see Chapter 30) stimu-
quite limited in comparison to the quantity of late rumen function and have been widely
metabolites secreted by the high-yielding ani- used in meat animals. The high-propionate
mal. Nutrient intake depends upon two fac- fermentations that result are advantageous to
tors: the amount of food dry matter eaten per a meat animal because they lead to conditions
day, and the concentration of nutrients within under which tissue growth is favoured.
that dry matter (nutrient density). Unfortunately, in dairy animals this leads to
an unacceptable reduction in the fat content of
milk and a diversion of nutrient resources
Rumen fermentation
towards body tissue rather than the mammary
The food intake of a ruminant is determined, gland.
in large part, by the ability of the rumen to One approach which has proved valuable is
degrade the feed quickly, and this depends the addition of live yeast (Saccharomyces cere-
upon the rate of fermentation. Diets contain- visiae) to the feed. This has been shown to
ing large amounts of soluble carbohydrate stimulate the ruminal breakdown of cellulose,
(either starch or sucrose) ferment rapidly, particularly in high-yielding animals that are
however they also lead to the development of receiving diets with a high content of
acid conditions in the rumen and the produc- digestible (chiefly ex-linked) carbohydrate. The
tion of lactate. These diets tend to have the improvements have been linked to two
highest nutrient densities (in terms of metabo- effects:
lizable energy per kg of food). The feeding of
• improved ability to buffer rumen pH so as
such diets produces a fermentation pattern in
to reduce the time during which it becomes
the rumen which is biased towards propionate
very add;
production and away from acetate (see
• lowered oxygen tension in the rumen con-
Chapter 16). Such conditions in the rumen
sequent on the yeast's ability to 'scavenge'
lead to a marked decrease in the proportion of
oxygen, resulting in conditions which
fat in milk. An increase in the molar percent-
favour the strictly anaerobic bacteria
age of propionate from 20-40% would be
responsible for cellulolysis.
expected to lead to a halving of the quantity of
fat in milk. In many countries there is a legal The low levels of fat in milk are caused by a
requirement for milk to contain a minimum reduction in the uptake by the mammary gland
content of fat (generally between 30 and 35 g of preformed fatty adds from blood. There is a
fat per kg milk). When high rumen propionate consequent increase in the uptake of these
levels are present, the milk may fail to meet adds by adipose tissue, leading to a repartition-
this minimum standard. ing of metabolites between milk secretion and
Manipulation of lactation 457
tissue growth. The effect of propionate appears deficiencies in amino acid in the protein start
to be mediated by insulin through changes in to exert a negative effect upon the composi-
the relative activities of the lipoprotein lipases tion of the supply to the mammary gland.
in adipose and mammary tissues. Work in a number of centres has shown that
increasing the supplies of lysine and methion-
ine can lead to increases in milk yield.
Protein supply
It is well accepted that the yield of milk in a
31.8.2 MANIPULATION OF MILK PRODUCTION
dairy cow is greatly influenced by the quanti-
BY EXOGENOUS HORMONES
ties of amino acids arriving at the mammary
gland through the blood. The first limiting It has been known for many years that admin-
amino acid is generally lysine, with methionine istration of bovine growth hormone leads to
the second. In ruminants there are two possi- increases in milk yield (section 31.7.1). This
ble sources of supply. One is the microbial pro- was of theoretical interest when the only
tein synthesized in the rumen, and the other is source of the hormone was its extraction and
the dietary protein which is undegraded in the purification from pituitaries removed at
rumen but which is digested in the small intes- slaughter. With the development of recombi-
tine (digestible undegraded protein, DUP, also nant gene techniques, it has been possible to
known as digestible by-pass protein). produce a synthetic product normally denot-
In ruminants at a low level of production, ed bST (bovine somatotropin). The hormone is
the quantity of microbial protein synthesized a protein and would therefore be degraded if
in the rumen will be sufficient to satisfy the given orally, for this reason it is administered
needs of the animal. Microbial protein is nor- daily by injection.
mally regarded as being a high-quality protein Typically, increases of between 25 and 40%
source in terms of its spectrum of amino acids. in milk yield may be expected in cows treated
As the level of animal production increases, so daily with bST and fed on diets with a rela-
too does the need for a supply of essential tively high energy content (see Figure 31.6).
amino acids from DUP. Where DUP repre- Part of the effect of bST is due to increases in
sents only a small part of the animal's amino the food intake of treated cows, but there are
acid supply, its composition is not very critical. also increases in the efficiency with which
Any deficiencies or imbalances in amino acid dietary energy is used for milk production. A
supply are masked by the contribution of further effect of bST is to change the partition
microbial protein. In the high-yielding dairy of nutrients so that there is a diversion of
cow, DUP may contribute over half of the resources away from tissue deposition into
needs for amino acids, and at this point any milk production.
45
Ci 40
:!. 35
:E
III
30
';' 25
~ 20
E 15
..... ..
'
~ 10
~ 5 bST Administration
0+-""-,-,,-,,,,-,-,.-,-,,-,-,,-,
2 6 10 14 18 22 26 30 34 38
Week of lactation
Figure 31.6 Typical effects of administering recombinant bovine somatotropin (bST) on milk yield in a cow.
MUSCLE AND MEAT 32
32.2 Biochemistry of muscular contraction 459
32.2.1 Structure of thick filaments 461
32.2.2 Structure of thin filaments 461
32.2.3 Mechanism of muscle movement 461
32.2.4 Control of muscle movement 462
32.3 Energy provision in muscle tissue 464
32.3.1 Myoglobin 465
32.4 Changes in muscle after death 465
32.4.1 Enzymes leading to the 466
degeneration of myofibrils
32.4.2 The role of myoglobin 466
32.4.3 Conditioning 467
32.4.4 Cold shortening 467
32.4.5 Effects of stress pre-slaughter 467
32.1 INTRODUCTION difficult to say whether each of these

envelopes actually represents a separate cell or
Much of animal agriculture is devoted to the
several cells; this distinction becomes impor-
production of meat for which skeletal muscle
tant in looking at processes of growth
provides the raw material. Although meat is
(Chapter 30).
derived from muscle there are important
Most of the space within the sarcolemma is
biochemical differences between the physio-
logically active tissue and the commercial occupied by the actual fibres, the myofibrils,
which are the distinctive operating mechanism
commodity, and changes to the carcass after
of muscle fibres. Each of the fibres can be seen
slaughter have to be carefully controlled to
as a series of repeating units known as sarcom-
ensure the provision of a hygienic product of
eres (Figure 32.1), at each end of which is a
high eating quality.
dark band known as the Z disk. Closer exami-
nation of the sarcomere shows that it is made
32.2 BIOCHEMISTRY OF MUSCULAR up of two different types of filament: at each
CONTRACTION end, and attached to the Z disk, are thin fila-
ments, whilst interdigitating between these are
The characteristic striated appearance of skele- the thick filaments (Figure 32.1b). The process
tal muscle is due to the arrangement of muscle of contraction in the muscle is illustrated in
fibres within the tissue. Within striated muscle Figure 32.1c, where it can be seen that the thick
it is rather difficult to define what is meant by and thin filaments slide past one another so as
an individual cell. The fibres of the muscle are to shorten the distance between the Z disks.
arranged within a plasma membrane called The thin filaments are predominantly made
the sarcolemma; each of these membranes up of the protein actin, whereas the major part
may contain many flattened nuclei so that it is of the thick filaments is myosin. This protein is
460 Muscle and meat
(a)
z z
.' 0 : T ',0 " t
(b)
(c)
Figure 32.1 Structure of muscle. The striated structure of muscle (a) is composed of a series of repeating
subunits (sarcomeres). Each sarcomere (b) has a Z disc at each end to which are attached the thin fila-
ments, t. Interdigitated between the thin filaments are thick filaments, T. The region of overlap between
the fibres is marked 0; contraction of muscle fibres is achieved by an increase in the degree of overlap
between fibres, (c).
known as myosin II, to distinguish it from muscle contraction is therefore to be found in

myosin I which is found in some cells which the relationship between the individual mole-
are motile but not muscular. The process of cules of actin and myosin.
Biochemistry of muscular contraction 461
32.2.1 STRUCTURE OF THICK FILAMENTS intervals along the fibre are the molecules of
the troponin complex; troponin T, troponin I,
Individual molecules of myosin are composed
and troponin C. Tropomyosin and troponin
of two heavy subunits (molecular weight
are integral parts of the mechanism that con-
about 200 kDa) and four light ones (molecular
trols the action of the muscle.
weight about 20 kDa). The heavy subunits
each have a globular 'head' and a long 'tail'
region; the tertiary structure of the polypep- 32.2.3 MECHANISM OF MUSCLE MOVEMENT
tide chain is almost completely that of an a-
helix. The tails of the heavy subunits are coiled The sliding action of muscle movement is
together (Figure 32.2a). Many of these individ- achieved by changes in the way in which
ual molecules polymerize to form a single myosin binds to actin. There are two important
heavy filament in such a way that all the glob- requirements for the process of movement:
ular regions are clustered at each end of the
• the binding between actin and the head of
filament (Figure 32.2b).
myosin is reversible and depends upon the
Purified myosin can be shown to have
presence or absence of ATP and ADP;
ATPase activity associated with the globular
• the shape of myosin, particularly of the
region. Myosin also has the ability to bind to
'head' region, can change depending on its
actin, the principal protein of the light fila-
chemical state.
ments within the myofibril.
The mechanism is illustrated in Figure 32.4.
The process starts with the individual 'head'
32.2.2 STRUCTURE OF THIN FILAMENTS
units of the myosin each lying close to a bind-
Actin is formed from a globular protein with a ing site on an actin molecule of a thin filament.
molecular mass of about 42 kDa which is At this time a molecule of ATP is bound to the
known as 'G actin'. This simple subunit aggre- globular region of the myosin. There is no
gates into long chains that come together as binding between the myosin and actin. The
pairs (F actin) and comprise the 'backbone' of movement starts with the hydrolysis of the
the light filaments (Figure 32.3), accounting for ATP to ADP and orthophosphate (Pi) which
about 67% of its weight. Actin bears binding both remain attached to the myosin. In this
sites on its structure that attach to complemen- combination the myosin binds firmly to the
tary sites on the globular part of myosin; the corresponding site on actin. Once the bond is
sites are all orientated in the same direction. formed, the myosin molecule changes shape
Attached along the length of the F actin by tilting the 'head' part and pushes the actin
chain is another protein, tropomyosin. At in one direction and the myosin in the oppo-
8.
b.
Figure 32.2 Structure of the thick filaments in muscle. A molecule of myosin (a) is composed of two
heavy subunits, each of which is composed of a long 'tail' region and a globular 'head'. The head units
also carry the light subunits which include the ATPase activity. Myosin molecules cling together to form
the thick filaments (b) with the tail units closely woven into a single strand and clusters of globular units
at each end.
462 Muscle and meat
Tropomysin
\ 2+ "\ 2+
Ca Actin Ca
•~
Troponin complex
LTnT Binds to Tropomyosin
\ ~ Tnt Binds to Bclin
TnC Binds to cJ+

Figure 32.3 Structure of actin. The main part of the molecule consists of two twisted strands (F actin),
each composed of a chain of globular (G actin) subunits. Associated with actin is the troponin complex
which controls the action of muscle in response to the presence or absence of Ca2+.
site one. After the movement, the bound ADP three linked proteins known as the troponin
and Pi are liberated and another molecule of complex. In the absence of Ca2 + the troponin
ATP is added to the myosin. The myosin is complex and tropomyosin together block the
now freed from the actin and the myosin binding sites on actin and prevent the interac-
'head' finds itself opposite the next binding tion with myosin.
site on the actin molecule. The cycle is now Within the muscle cell, the supply of Ca2 + is
ready to start again. regulated by a series of internal membranes
known as the sarcoplasmic reticulum, which
concentrate and bind it in combination with
32.2.4 CONTROL OF MUSCLE MOVEMENT
the protein calsequestrin until required. The
Obviously, muscle activity must be one of the calcium concentration in the sarcoplasmic
best coordinated and most closely regulated of reticulum is greater than 10-3 M. The calcium is
all processes. The movement of muscle must held within vesicles known as calciosomes.
therefore be under the control of a series of The trigger for muscle movement is the release
very precise mechanisms. Additionally, mus- of Ca2 + from this system via a protein channel,
cle tissue has an important role in heat pro- ryanodine (see section 32.4.5).
duction which is not related to coordinated There are always two important parts to
movement. any control system - the part that turns it on
The physiological trigger to muscle contrac- and the part that turns it off. In the absence of
tion is Ca2 + which changes the interaction of any stimulus to muscle movement, the supply
myosin and actin. The role of Ca2 + is mediated of Ca2+ in the cell is rapidly reduced to less
through the two other groups of proteins in than lO-6M by Ca2 +-ATPase similar to Na+-K+
the thin filament: tropomyosin, which is a ATPase. The system moves two Ca2 + ions for
long fibrous protein running alongside the every ATP molecule that is hydrolysed. This
double filament of actin; and an assembly of use of ATP in the removal of the muscle stim-
Biochemistry of muscular contraction 463
Stages In the
Thin filament movement cycle In
muscle
1
Thick filament 1. ATP attached to
Myosin Is hydrolysed
by myoaln ATPaae.
DP" P, 2. ADP and PI remain

aHached to myosin.
1 3. Myosln-AOP-P.
complax blnda firmly
to aita on actin.
1 4. Conformational
change in myosin mova.
myoain in ,elation to
...
actin.
5. ADP and ~ are

IIbarated 'rom myoaln.
~
1
ATP ___ New molecula of ATP ia
ADP + PI bound to myosin
allowing myosin to be
raleasad from
a«achment to actin.
ATP
Figure 32.4 The muscle contraction cycle.
ulus adds further to the amount of energy that through the muscle cell as a series of tubules
is required for muscular work. arranged at right angles (transverse) to the
The stimulus to Ca2 + release from the sar- muscle fibres in the region of the Z disks. This
coplasmic reticulum is transmitted by the gives the cell the ability to transmit the depo-
depolarization of the outer cell membrane at larization of the outer membrane, in response
the junction between nerve and muscle cells. to incoming nervous signals in positions
The outer membrane (sarcolemma) of muscle which are physically close to the operating
cells has specialized zones, which run into and areas of the fibres.
464 Muscle and meat
32.3 ENERGY PROVISION IN MUSCLE TISSUE esterified fatty acids (NEFA) and plasma glu-
cose. They are richly supplied with capillaries
It is a common observation that all muscles are
to supply the large amounts of substrates that
not of the same colour; this is particularly easy
they need. The pattern of metabolism is pre-
to see in the carcass of a chicken, where the
dominantly aerobic oxidation, leading to a
muscle of the breast is pale pink whereas the
high oxygen demand within the cytochrome
meat of the legs is a much redder shade. On
system of the mitochondria. This need is met
cooking these yield meats which are light and
by the action of myoglobin, an oxygen carrier
dark, respectively. The different colours are
within the cytosol of the fibres. When resting,
related to the functions of the different muscle
the major fuel source (about 80%) for the slow-
areas and to their biochemical and physiologi-
twitch fibres is NEFA. As the physical demands
cal properties. Three different types of fibre
on the muscle increase during exercise, the
are commonly recognised: two of these are
proportion of energy that can be derived from
white and one red. Within any particular mus-
this source drops and more glucose is used. At
cle it is common for all three types of fibre to
the highest levels of muscle output, the princi-
be found, although the predominant one will
pal energy substrate is glucose.
differ. Basically, the red fibres are found in
Phosphocreatine is used particularly in the
muscles that have a high and persistent level
white fibres as a reservoir of group transfer
of physical activity. The red fibres are denoted potential. Phosphocreatine and ADP have a
as type I whereas the white are type II, a cate- reversible relationship with creatine and ATP
gory which is further split into IIA and lIB under the influence of the enzyme creatine
depending upon the type of oxidative metab- kinase (Figure 32.5).
olism that predominates. All types of muscle Phosphocreatine brings advantage to mus-
share a common contractile mechanism and a cle because, although it is a nearly instanta-
consequent need for ATP. Where they differ is neous source of ATP, it does not use up the
in the fuel which supplies their ATP. cell's supply of adenosine. If the muscle cell
There are four potential fuels that can pro- stored energy only in the form of ATP, very
vide the ATP for muscular activity: soon all of the adenosine would be in the form
• phosphocreatine of ATP and none in ADP or AMP. This would
lead to the inhibition of a wide range of
• glycogen
• plasma glucose allosteric enzymes (e.g. phosphofructokinase)
• plasma lipids. and a halt to substrate use and respiration.
Muscle glycogen acts as a reserve of energy
The importance of individual fuels varies in within muscles. The amount stored is quite
the different types of muscle fibre and may small but is able to provide energy for muscles
also change with circumstances. that are not in persistent activity. These tend
The characteristics of the three type of mus- to be the white, fast-twitch fibres and, not sur-
cle fibre are summarized in Table 32.1. The red, prisingly, these have much higher capacity for
type I fibres have a slow twitch rate after stim- gycogenolysis and glycolysis. There are two
ulus (they are often referred to as 'slow-twitch' families of fast-twitch cells (A and B) which
fibres). The motor neurones of this type of differ in the ways in which they are able to uti-
muscle have a very low threshold of activation lize their supplies of glycogen. Type B fibres
which means that these fibres are the ones have high glycolytic ability but a low potential
which will be activated first. These are pre- for mitochondrial oxidation, they must there-
dominantly fibres which gain their ATP from fore gain their energy by lactate production
mitochondrial oxidation of substrates that are and recycling of glucose through the Cori
taken up from blood, in particular plasma non- cycle (see Chapter 16). Type A fibres can oxi-
Changes in muscle after death 465
Table 32.1 Differences between muscle fibre types
Fibre type
Property Type I Type II A Type II B
Colour Red White White

ATPase activity Low High High
Creatine kinase Low High High
Glycolytic enzymes Low High High
Mitochondrial oxidation High High Low
Myoglobin High Low Low
Twitch speed Slow Fast Fast
Fatigue Resistant Resistant Fatiguable
dize glycogen completely to COz and water, The sixth is the one that can be occupied by a
thereby increasing their ATP yield from each pair of electrons from a molecule of oxygen.
of the glucose units of glycogen by about 18
times. The type B fibres are easily fatigued
32.4 CHANGES IN MUSCLE AFTER DEATH
because their energy supply is, in the short
term, non-renewable. Immediately after death, the oxygen supply
to the muscle is cut off and conditions become
anaerobic, so that there is no longer a supply
32.3.1 MYOGLOBIN
of ATP from oxidative phosphorylation. Even
Myoglobin shares the same haem structure in those muscles that normally operate under
(iron protoporphyrin IX) with haemoglobin, anaerobic conditions through the action of the
although unlike haemoglobin it exists as a Cori cycle (Chapter 16), the ability to recycle
monomeric protein structure with a single lactate/glucose through the liver is lost due to
polypeptide chain of 153 amino acids. Unlike the cessation of blood circulation. The imme-
haemoglobin, myoglobin is purely a carrier of diately available energy sources in muscle,
oxygen and does not transport COzor H+. The ATP and creatine phosphate, are used up in a
presence of the haem within the protein struc- very short time. Thereafter the supply of ATP
ture gives the characteristic red colour to mus- must come from the anaerobic oxidation of
cle. the limited supplies of glycogen to lactate
The iron atom is held into the structure of (Chapter 16). The demand for energy is not
the porphyrin ring as a coordination com- completely lost at death: the Caz+ ATPase con-
pound. Iron has a coordination number of 6, tinues to maintain the Caz+ gradient in the
and of these bonds, four are with the nitrogen sarcoplasmic reticulum and uncoordinated
atoms in the individual five-membered rings muscle twitching can be observed.
of the porphyrin. The fifth is with a nitrogen The actin and myosin components of the
atom in the side chain of a histidine group muscle fibre are only able to separate when
(amino acid 92 out of 153) in the polypeptide. ATP is bound to myosin, and as supplies of
H COOH oI HI COOH
ATP + I I
H-N-y-CH2 ~
....- ADP +
I
O--P""'-N-C-CH
T 2
a NH
I
NH Creatine
kinase
Creatine Phosphocreatine
Figure 32.5 Creatine kinase catalyses the reversible transfer of phosphate groups from ATP to creatine.
466 Muscle and meat
ATP become exhausted the overlapping actin 32.4.1 ENZYMES LEADING TO THE
and myosin become strongly bonded together DEGENERATION OF MYOFIBRILS
and are thus firmly cross-linked. This results in
The changes in myofibril structure require the
the muscle becoming very hard and the presence of Ca 2 + ions; if these are removed by
process known as rigor mortis takes hold.
the use of a complexing agent (such as EDTA)
Maximum rigor occurs at about 24 hours after then only limited degeneration takes place. It
death, depending upon both temperature and is interesting to see that this is a further role for
the species of the animal.
calcium which is so closely involved in the
Prior to rigor, muscle is quite extendable
process of muscle contraction. The e~zy~e
under load and acts like a spring, returning to
responsible for myofibrilar degradatIon tS
its original length when the load is removed.
known as calcium-activated factor (CAP) and
After the cross-linking of the filaments this
has been prepared from a wide range of
elasticity is lost. On cooking, meat taken from
species. Its distribution is not limited to .muscle
an animal before rigor mortis is relatively ten-
and it can be prepared from many dtfferent
der. However, it becomes extremely tough as
tissues.
the actin and myosin filaments link together.
CAF specifically hydrolyses troponin T and,
In order for muscle to be converted into
to a lesser extent, troponin I. It can also trigger
meat it has to go through a process of condi- the release of a-actinin (a protein which binds
tioning where it is allowed to mature for a
to actin within the Z disk). Another protein of
period of days or even weeks before co~ the Z disk, desmin, is also degraded by CAF,
sumption. During this period the meat will further weakening the attachment of the thin
gain greatly in tenderness and possibly ~n fibres to the disk.
flavour. Perhaps surprisingly, the changes m Within the living animal, most cellular com-
tenderness are not due to a decrease in the
ponents are constantly undergOing break-
amount or quality of collagen in the muscle, down and resynthesis. One of the mecha-
which tends to remain constant during con-
nisms for breakdown involves the activity of
ditioning. Muscle with a high level of colla-
the cell's lysosomes which act very much as
gen, particularly that from old animals, leads
intracellular stores of a whole range of
to the formation of meat with a high level of
hydrolytic enzymes. Amongst those responsi-
collagen. This gives one biochemical reason
ble for protein degradation are collagenase
for the fact that meat from older animals
(breaking down collagens), peptidases (acting
tends to be tough.
on smaller peptides) and a whole range of pro-
The main changes in conditioning occur
teolytic enzymes known as the cathepsins.
within the fibres of the sarcomeres. The area
Although their levels are much higher in liver,
around the Z disk of each fibre (where the thin
two of the cathepsins, Band 0, are found in
filaments are attached) starts to break down.
striated muscle. Both of these enzymes are
The thin filaments are thus free to pull away.
able to catalyse a limited breakdown of actin
Meat that has been conditioned for about 3-4
and myosin to smaller subunits.
days will be as extendable under load as mus-
Experiments on muscle from cattle have
cle from a freshly killed animal; however, once
indicated that the activity of CAP is more
the applied load is removed the fibres will not
important than that of the cathepsins in meat
contract back to their original size. These
conditioning.
changes imply a gradual breakdown of pro-
tein structure within the myofibrils. They
32.4.2 THE ROLE OF MYOGLOBIN
occur under the influence of enzymes from
within the muscle itself rather than from any Transport of oxygen within the extended cell
bacterial fermentation. structure of the sarcomere requires that mus-
cle has its own intracellular carrier system. ruminants, cattle, sheep and goats, take similar
Myoglobin can exist in three different forms lengths of time (approximately 7-10 days) to
dependent upon the structure of the haem achieve maximum tenderness, whereas pig
group. At its heart, the haem molecule has an meat requires a shorter period and chicken
atom of iron attached to the centre of the por- may require only a few days.
phyrin ring. The iron is usually in oxidation
state II (ferrous), and is attached covalently to
32.4.4 COLD SHORTENING
a nitrogen atom on the side chain of a histidine
residue in myoglobin's polypeptide chain. A complete carcass, particularly of a beef ani-
Oxygen attaches directly to the iron atom to mal, takes a long time to cool down after
form oxymyoglobin; the configuration with- death. Some of this is due to the poor thermal
out oxygen is known as deoxymyoglobin. In a conductivity of the thick pieces of flesh, but a
third and physiologically inactive variant the further factor, heat production in the carcass is
iron atom is oxidised to the III state (ferric). that, due to ongoing biochemical reactions,
After death the oxygen attached to myoglo- may continue for a considerable period. If the
bin is used up very rapidly, and the molecule temperature of the carcass is reduced too
assumes the reduced form of deoxymyoglo- quickly to assist in the maintenance of
bin, which has a very dark, almost purple hygiene, the process known as cold shorten-
colour. On cutting meat, the myoglobin picks ing results.
up oxygen, forming the bright red oxymyoglo- The contraction of muscle is controlled by
bin. This is the colour that consumers associate the levels of free Ca2 + within the fibre: normal
with fresh meat and one which retailers often control mechanisms ensure that levels of Ca 2 +
try to accentuate with appropriately tinted are maintained at low levels as long as the mus-
lighting. If meat is left exposed to the atmos- cle does not receive neural stimuli. Rapid chill-
phere for extended periods the haem group ing of muscle in the early post-mortem period
oxidizes from iron (II) to iron (III), and this fer- can lead to an increase in intracellular Ca2 + at a
rimyoglobin is of a brown colour (also referred time before the cell's ATP resources have been
to as metmyoglobin). The colour development depleted. Under these conditions the thin and
is not a simple event - at depth within thf' thick filaments move together, increasing their
meat, the mitochondrial cytochrome system is overlap so that the muscles take on their most
able to reduce ferrimyoglobin back to oxymyo-
contracted form. In extreme cases their length
globin. This may also permit limited aerobic
may reduce to 40% of the resting state. With the
metabolism to continue within the meat.
onset of rigor mortis, cross-linking of thick and
thin filaments becomes fixed. Meat in which
32.4.3 CONDITIONING
the muscles have become shortened is tough,
The process of conditioning is central to the and even after extended conditioning does not
production of meat from a newly killed carcass become as tender as normal meat. The effect of
and can make a very great deal of difference to chilling on Ca 2 + levels is probably due to an
the eventual quality of the product. The tem- impairment of the ability of the sarcoplasmic
perature of conditioning is an important com- reticulum to bind the ion and possibly also to its
ponent of the process. If the carcass is allowed being released during the post-mortem degener-
to achieve too high a temperature then the risk ation of mitochondria.
of bacterial contamination of the product
becomes serious. The principal improvement
32.4.5 EFFECTS OF STRESS PRE-SLAUGHTER
during conditioning is in the tenderness,
although in some species there may also be One of the main effects of animal stress,
changes in flavour. Meats from the major whether caused by physical exhaustion, rough
468 Muscle and meat
Valine Valine
-----t-- -----t--
----r--
AlanirllJ .A!an:ne
----t--
---t---
Valine Valine
---t--
.
----t-
Arginine
_615 ----r-
Cysteine
_615
Serine Senne
-----t--
Asparagine
-----~--
Asparagine
-----J--
G!ycine
-----~--
Glycine
Figure 32.6 Normal (left) and mutated (right) gene responsible for malignant hypothermia (porcine stress
syndrome). A single nucleotide substitution (T for C) leads to the incorporation of cysteine instead of
arginine in the calcium channel protein, ryanodine.
handling, transport, fear, severe climatic condi- duce lean carcasses. It was found that in sever-
tions or hunger, is to increase the levels of cate- al breeds there were family trees of animals
cholamine hormones produced in the adrenal with a genetic abnormality such that quite mild
medulla. In turn this leads to elevation of the stress may be fatal. In most species the problem
activity of intracellular protein kinases and the is known as malignant hypothermia, but in
activation of glycogen phosphorylase. Stressed pigs it is called porcine stress syndrome (PSS).
animals therefore have reduced levels of glyco- The clinical findings associated with the condi-
gen in muscle, leading to a reduced capacity for tion included raised intracellular calcium levels
lactate production (see Chapter 16) and a final which increase muscle glycogenolysis (and
pH of above 5.9 in the meat. The result is that in therefore heat production) and muscle tone.
some muscles, but not all, the meat is darker The lesion is a biochemical one at the level of
and may be slightly firmer and drier (DFD the gene which codes for ryanodine, one of the
meat: dark, firm, dry) than that from nOn- proteins of the calcium channel that releases
stressed animals. In beef animals the muscles calcium from the sarcoplasmic reticulum.
most affected tend to be those of the hindquar- There is a simple substitution of a thymine
ter which are normally the most expensive cuts. residue for a cytosine at base 1843 in the DNA
A further problem with a very definite bio- strand that codes for the protein, which
chemical basis is to be found in particularly changes the triplet codon from CGC to TGC
stress-prone breeding lines of pigs. This prob- (Figure 32.6). The amino acid that is coded by
lem is not confined to pigs but is also found in CGC is arginine, but TGC leads to cysteine
many species of livestock and even in man. It incorporation into the polypeptide. There are
has perhaps been made more obvious in pigs other abnormalities in the base sequence of this
through the intensive breeding and monitor- gene in stress-susceptible pigs, but these do not
ing schemes introduced to produce animals lead to variations in the amino acid which is
that grow quickly and economically, and pro- coded for. For instance, base 3942 codes for
thymine in normal pigs and cytosine in sus- elimination by selective breeding away from
ceptible ones. This leads to a change in triplet affected lines. After death, affected animals
codon from GGT to GGc. Both of these codes which have survived the stresses of transport
are valid combinations for glycine so that there and pre-slaughter handling produce carcasses
is no change in the eventual polypeptide with serious problems. Due to the elevated
formed. intracellular calcium levels, rigor mortis within
The change from the normal arginine to major muscle groups sets in almost immedi-
cysteine leads to the formation of an abnormal ately, there is a very rapid production of lac-
variety of ryanodine, which is far more sensi- tate, and the pH drops to 5 or below. The
tive to the stimuli leading to Ca 2 + release. Pigs muscles most affected are those yielding the
which suffer from this problem were found to high-value cuts of meat in the back and legs.
be extremely sensitive to anaesthetics, partic- The meat produced from these animals tends
ularly halothane, and this has been used as a to be unusually pale and soft, and oozes fluid
very simple way of distinguishing these anion cutting. Such meat is termed pale, soft,
mals. As the condition is due to a gene abnor- exudative (PSE) pork.
mality, much progress has been made in its
FURTHER READING
Alberts, B., Bray, D., Lewis, J., Raff, M., 3rd edn, Blackwell Scientific Publications,
Roberts, K. and Watson, J.D. (1989) Oxford.
Molecular Biology of the Cell, 2nd edn, Gale E.S. (ed.) (1981) The Molecular Basis of
Garland Publishing Inc., New York. Antibiotic Action, 2nd edn, John Wiley,
Allen, J.C and Hamilton, R.J. (eds) (1989) London.
Rancidity in Foods, 2nd edn, Elsevier Applied Gunstone, F.D., Harwood, J.L. and Padley,
Science, London and New York. F.B. (1994) The Lipid Handbook, 2nd edn,
Anderson, J.W. and Beardall, J. (1991) Chapman & Hall, London.
Molecular Activities in Plant Cells, An Gurr M.1. (1992) Role of Fats in Food and
Introduction to Plant Biochemistry, Blackwell Nutrition, 2nd edn, Elsevier Applied
Scientific Publications, Oxford. Science, London.
Bender, D.A. (1992) Nutritional Biochemistry of Gurr, M.1. and Harwood, J.L. (1991) Lipid
the Vitamins, Cambridge University Press, Biochemistry, An Introduction, 4th edn,
Cambridge, UK. Chapman & Hall, London.
Buttery, P.J., Boorman, K.N. and Lindsay, D.B. Horton, R.H., Moran, L.A., Ochs, R.S., Rawn,
(eds) (1992) The Control of Fat and Lean J.D. and Scrimgeour, J.G. (1993) Principles of
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Campion, D.R., Hausman, G.J. and Martin, R.J. Kaneko, J.J. (ed.) (1989) Clinical Biochemistry of
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Plenum Press, New York. London.
Christie W.W. (ed.) (1981) Digestion, Kigel, J. and Galili, G. (1995) Seed Development
Absorption and Transport of Lipids in and Germination, Marcel Dekker,
Ruminant Animals, in Lipid Metabolism in Philadelphia.
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Christie W.W. (ed.) (1981) Lipid metabolism in 2nd edn, Longman Scientific and Technical,
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21-56. Biochemistry and Molecular Biology, John
Davis P.J. (ed) (1995) Plant Hormones: Wiley and Sons Ltd, Chichester.
Physiology, Biochemistry and Molecular Lehninger, A.L., Nelson, D.L. and Cox, M.M.
Biology, 2nd edn, Kluwer, Dordrecht. (1993) Principles of Biochemistry, 2nd edn,
Dennis, D.T. and Turpin, D.H. (eds) (1990) Worth, New York.
Plant Physiology, Biochemistry and Molecular Machlin L.J. (ed) (1991) Handbook of Vitamins,
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472 Further reading
Mathews, C.K. and Van. Holde, K.E. (1996) in Meat Research, Volume 7, Elsevier Science,
Biochemistry, 2nd edn, Benjamin/Cummings, London.
Menlo Park, California. Primrose, S.B. (1991) Molecular Biotechnology,
McDonald, P., Edwards, RA., Greenhalgh, Blackwell Scientific Publications, Oxford.
J.F.D. and Morgan, c.A. (1995) Animal Roberts, J.A. and Hooley, R. (1988) Plant
Nutrition, 5th edn, Longman Scientific and Growth Regulators, Blackie and Son Ltd,
Technical, Harlow, Essex. Glasgow and London.
McDowell, L.R. (1989) Vitamins In Animal Ruckebusch, Y., Phanent, L.P. and Dunlop, R
Nutrition, Comparative Aspects to Human (eds) (1991) Physiology of Small and Large
Nutrition, Academic Press, San Diego, Animals, Marcel Dekker, Philadelphia.
California. Salisbury, F.B. and Ross, C.W. (1992) Plant
Mepham, T.B. (1987) Physiology of Lactation, Physiology, 4th edn, Wadsworth Publishing
Open University Press, Milton Keynes. Co., Belmont, California.
Mertz, W. (1987) Trace Elements in Human and Van Soest. P.]. (1994) Nutritional Ecology of the
Animal Nutrition, 5th edn, Academic Press, Ruminant, Cornell University Press, Ithaca,
New York. New York.
Pearson, A.M. and Dutson, T.R (eds) (1991) Yeagle, P. (1987) The Membranes of Cells,
Growth Regulation in Farm Animals, Advances Academic Press, San Diego, California.
INDEX
abscisic acid (ABA), 387-8 agricultural waste, treatment, 211-12

biochemistry, 387 Agrobacterium tumefaciens
physiological activities and applications, 387-8 biosynthesis of auxins, 385, 386
synthesis and transport, 387 crown gall disease, 384
abscission, see plant growth and development vector for DNA, 311
absorption of nutrients, ruminants/non-ruminants, Alar, see daminozide
395-411 albumins, properties, 67
ACC (amino cyclopropane carboxylic acid), 388-90 alcohol, see ethanol
ACC synthase, 388-90 aldolase, 143
ACC oxidase, 388-90 alkali disease (blind staggers, selenium toxicity), 52-4
acetals alkaline tide, 406
structure, 22 alkalis, defined, 13
sugar acetals, formation, 24--{) alkaloids, 55-7
acetate structures, 56
formation from glucose in digestive systems, 209-10 allopurinol, enzyme inhibitor, 84, 85
role in fatty acid synthesis, 252-3 allosteric enzymes, 90-1
acetic acid, dissociation, 13-15 allosteric regulation, feedback inhibition, 323
acetone, ketotic animals, 181-2 kinetics, 91
acetoxy acid synthetase, herbicide inhibition, 83 structure, 92
acetyl-CoA a-amylase, starch breakdown, 340, 341
condensation with oxaloacetate, 151 a-ketoglutarate, conversion to succinyl-CoA, 152
synthesis, 149-50, 253 a-oxidation of fatty acids, 182-3
conversion to malonyl-CoA, 253-6 a-tocopherol, 48, 125, 185-6, 227
role in synthesis of long-chain saturated fatty acids, a-tocotrienol, 125, 185
256--61 amino acid-activating enzymes, 297-8
acetyl-CoA carboxylase, control of activity, 254--{) amino acids, 51-70
N-acetyl-glucosamine, structure, 25 absorption, mechanism, 409
acetylcholine, transfer of nerve impulse, 82 activation, 297-9
acid-base catalysis, enzyme activity, 74 aliphatic synthesis, pathway, 288
acids, defined, 14 ammonia excretion, 197
actin, role in muscle, 462 aromatic, 284-5
actin filaments in cytoskeleton, 9 biosynthesis, 284-8
acyl carrier protein inhibition by herbicides, 83, 289-90
function in fatty acid synthesis, 256 branched-chain aliphatic, 285-7
structure, 257 breakdown, 194-9
acylglycerols, 39-42 deamination, 195
adenine, 96, 98 essential
adenosine di/triphosphate see ADP; AIP for non-ruminant animals, 289
adipocytes content in fish meal and groundnuts, 138
fat deposition, 430-1 content in barley proteins, 339
precursor cells, 429-30 N-terminal, effects on half-life of proteins, 417-18, 420
adipose tissue, 10 non-protein
brown, see brown adipose tissue and related compounds, 52-7
growth, 429-31 structures, 54
ADP, 87-90 nutritional role, 287-9
ADP/AIP, oxidative phosphorylation regulation, 169-71, oxidation of carbon skeletons, 195-7
325 pathways, 196
adrenaline, effect of, 325, 328, 431 precursor functions, 199
adrenaline, structure, 437 selenium-containing, 52-4
474 Index
shikimic acid pathway, 284-6 biochemistry, 374-5
structures, 52-3 physiological activities and applications, 377-9
sulphur-containing, 53 synthesis and transport, 376
supply to mammary gland, 451 synthetic, 375-7
synthesis, 279-90 avoparcin, non-ionophore, 442
transamination reactions, 194-5
amino acyl tRNA, formation, 300 bacteria
amino acyl tRNA synthetases, 297-8 cell wall, 6, 444
p-aminobenzoic acid, see PABA fatty acid synthesis, chain length specificity, 260
l3-aminopropionitrile, 54, 55 bacteriophorage, 310-11
ammonia assimilation, 282-4 Gram-negative/positive, 6, 442-444
ammonia excretion, 197 bases, defined, 14
amprolium, structure, 107 beer and whisky production, 341-2
amygdalin, 33 behenic acid, 37
amylase, starch breakdown, see a-amylase beta-agonists, see l3-agonists
amylopectin, structure, 27 l3-agonists, 435-8
amylose, structure, 28 effects on meat-producing animals, 436-7
anaerobic environments, 207 growth manipulation, 436-8
androgens, see oestrogens and androgens mode of action, 437-8
anthocyanins,58-61 beta-carotene see (13-)carotene
antibiotics beta-oxidation, see l3-oxidation
growth promoters, 440-5 l3-oxidation, 173-82
ionophores, 440-3 odd-numbered acids, 176-8
non-ionophores, 443-5 peroxisomes and glyoxisomes, 179-81
effects on peptidoglycan synthesis, 444 unsaturated acids, 17S-9
effects on protein synthesis, 102 biotin, 113-14
antibodies, 91-3 deficiency, 111
immunoglobulins, 92-3 birds, uropygial gland, branched-chain fatty acid produc-
structure, 92 tion, 260
antioxidants blind staggers, 52-4
synthetic, structures, 187 blood, colligative properties, 18
vitamin E, 124-5, 185 blue-green algae, 3
antisense RNNgenes, 310, 391 body composition, 134
antiport transport systems, 316 body temperature, heat production, 413-14
apical dominance, see plant growth and development bone
arabinogalactans, 32, 344 function, 10
arabinose, 21 growth,427-8
arabinoxylans, 29, 31 structure/calcification, 42S-9
arachidic acid, 37, 38 see also skeletal growth
arachidonic acid, 37, 38 boron, plant nutrition, 363
aromatic amino acids, 284-5 bovine growth hormone, 457
biosyntheSis, pathway, 286 bovine somatotropin, 457
Arrhenius equation, 79 effects on milk yield, 457
ascorbic acid, structure, 118, see also vitamin C bovine spongiform encephalopathy (BSE), 69-70
assimilate movement, see plant growth and development brain and nervous tissue, 10
atmosphere, effects on plant growth, see plant growth brassicas, 348
and development brazil nut, 339
ATP, 87-90 bromalain, 74
complex formation with magnesium, 128-9 brown adipose tissue, 117, 419
phosphate group transfer, 90 browning reactions, 352
structure, 88 BSE, see bovine spongiform encephalopathy
ATP production, glycolysis and TCA cycle, 154 BST, see bovine somatotropin, 457
ATP synthesis buds, dormancy, see plant growth and development
chemiosmotic model, 166-8 butyrate
electron transport coupling, 166-8 formation in digestive systems, 210, 212, 39S-9
Flo-ATPase, 168 utilization for milk production, 453
photosynthesis, 225-6
ATP-dependent ion pumps, 317-20, 415-16 C4 plants, photosynthesis, 232-3
auxins, 374-9 CAF, see calcium-activated factor
Index 475
calcium, 127-8 cassava, 352
metabolism, abnormalities, 128 castration effects, 433-4
milk fever, 128 catecholamines, 435-9
osteomalacia, 128 increase in pre-slaughter stress, 468
osteoporosis, 128 mode of action, 437-9
plant nutrition, 362 cattle
rickets, 128 body composition, 135
calcium-activated factor, 466 mineral composition, 135
callose, 248-50 CCc, 382
callus, differentiation, see plant growth and development cDNA synthesis, 308-9
Calvin cycle, 218, 226-9, 230 cell compartments, 3-10, 313
diagrammatic representation, 227 cell division, differentiation of callus, see plant growth
cambium, growth and differentiation, see plant growth and development
and development cell membrane, semipermeable properties, 17-18
c~P,321,344,438 cell structure and function, 3-10
campesterol, 47, 49 animal model, 5
camphor, structure, 46 cell walls, 6
Canavalia ensiformis Gack bean) seeds, canavanine, 52 chloroplast, 4, 8
canavanine, 52,54 cytoplasm, 4--6
canola, see oilseed rape cytoskeleton, 4, 8-9
carbohydrates, 19-34 endoplasmic reticulum, 4, 6-7
digestion flagella and cilia, 4, 9
monogastric animals, 396-8 glyoxysomes, 4, 7-8
ruminants, 398 Golgi bodies, 4, 7
empirical formula, 19 lysosomes, 4, 7
gross energy, 133 mitochondria, 4, 8
metabolism, control, 327 nucleoid and nucleus, 4, 6
in plant nutrition, 137-8 peroxisomes, 4, 7
protein, attachment of, 66 plant model, 5
seeds, 336-7 plant vacuoles, 4, 7-8
storage,26-9,133-4,336-7 plasma membrane, 3-4
synthesis, 226-9, 237-50 ribosomes, 4, 6
in temperate and tropical grasses, 137-8, 347-8 specialization and interaction, 9-10
carbon assimilation, plant nutrition, 226-9, 364 vacuoles, 7-8
carbon dioxide levels cell walls, see plant cell walls
effects on photosynthesis, 372 cellobiose, structure, 26
effects on plant growth and development, 370-1 cellular retinol-binding proteins (CRBPs), 121
carbon fixation, 226 cellulose
amounts, global, 217 properties, 29
carbon and nitrogen metabolism structure, 30
interaction, 363--6 synthesis, 248-50
senescence and nutrient cycling, 366 ceramides, 270-1
carcass cereal proteins, 337-9
anaerobic catabolism, 213-14 amino acid composition, 337-8
catecholamine hormones, increase in pre-slaughter solubility classification, 337-8
stress, 468 cereals, lodging control, 382
mineral composition, 134--5 cerebrosides, 44, 270-1
and skeletal muscle, 465--69 cerebrospinal fluid, colligative properties, 18
carnitine chaperone protein, 301-3
biochemical functions, 119, 174--6 chemiosmotic model of AIP synthesis, 162
deficiency, 111 chickpea, lathyrogens, 55
carotenoids, 45, 219 chilling injury, see plant growth and development
~-carotene, 219 chloride
metabolism, 121 concentration in mammalian cell, 129
structure, 48, 120 plant nutrition, 363
role in photosynthesis, 219-20 chlorogenic acid, 57, 352
xanthophylls, 219 chlorophylls
carvone, structure, 46 photosynthesis, 218-19
casein, 68,451-2 structure, 220
476 Index
chloroplasts, 4, 8 structure, 33
photosynthesis, 218-19 cyclo-oxygenase, 188-9
protein synthesis, 306-7 cysteine proteases, 74
structure, 219 cytochrome b-f complex, 222, 225
cholecalciferol, see vitamin D3 cytochrome b, c1 complex, electron transport chain, 165
cholesterol, 47 cytochrome oxidase, electron transport chain, 165--6
absorption from small intestine, 403 cytochromes, 163
structure, 49 cytokinins,384-7
synthesiS, 276-7 biochemistry, 384
choline, 118-19 physiological activities and applications, 384-7
deficiency, 111 synthesis and transport sites, 384
chromatin, composition, 99 cytoplasm, 4-6
chylomicron formation, 403 cytoplasmic streaming, 9
chymotrypsin cytosine, 96, 98
mechanism, 74-5 cytoskeleton, 4, 8-9
digestion, 408
cilia, 4, 9 dairy products, fermentation, 213
cimaterol, structure, 437 daminozide, growth retardants, 382-4
cinnamic acid, 58 dark, firm, dry (DFD) meat, 468
citrate (citirc acid) dark reactions, photosynthesis (Calvin Cycle), 226-9
conversion to isocitrate, 151 dehydroascorbic acid, structure, 118
in fatty acid synthesis, 254, 255 Delphinium spp, alkaloids, 57
structure, 354 deoxyribonucleic acid, see DNA
citric acid cycle, see tricarboxylic acid cycle deoxyribonucleotides, synthesis, 292
Claviceps spp, 55 desaturation
clenbuterol, structure, 437 fatty acids, 262-3
CO 2 production, TCA cycle, 150 polyunsaturated fatty acids, metabolic transformation,
cobalt, 130 264
vitamin B12, 115--17 development, see growth
coconut oil, 35, 40 DFD, see dark, firm, dry (DFD) meat
coenzyme Q, 45, 48, 164 DHA, see docosahexaenoic acid
coenzymes, 84-90 dicots, hemicelluloses, 29
redox and biological oxidations, 84-7 lignin, 57
cold requirement, see plant growth and development dicoumarol, structure, 125
cold shortening, muscle tissue after death, 467 digalactosyldiacylglycerols, 43--4, 268-9
collagen, 66,117 digestion, 208-10, 395--411
growth, 425--7 carbohydrate, 396-8
Colletotrichum circinans, 57 fermentation pathways and VFA production, 208-10
concanavalin A, 34 lipid, 399-406
conditioning, muscle tissue after death, 467 protein, 406-10
copper, 130 structure and function of digestive tract, 10, 395--6
deficiency, 130 dihydrophaseic acid. 387-8
in plant nutrition, 363 disaccharides, 25--26, 243--6
Cori cycle, glucose regeneration, 208, 209 lactose synthesis, 243--5
correlative effects, growth retardants, see plant growth structure, 25--6
and development sucrose syntheSis, 245--6
p-coumaric acid, 58 synthesis, 25
covalent intermediate formation, enzyme-substrate com- dissociation constants and pK. values, common organic
plex,74-6 acids, 16
covalent modification, metabolic regulation, 323--5 diterpenoid alkaloids, 57
crassulacean acid metabolism, 233--4 DNA, 96-100, 303--6
diagrammatic representation, 235 cDNA synthesis, 308-9
creatin kinase, catalytic transfer of phosphate from ATP double helix, 98-9
to creatine, 465 hydrogen bonding, 97-8
Creutzfeld-Jacob syndrome, 69 manipulation, 308-11
crown gall disease, Agrobacterium tumejaciens, 384 organelle, 100
i3-cyanoalanine, 54, 55 replication, 292-6
cyanocobalamin, see vitamin BI2 sequencing, 308
cyanogenic glucosides, 33--4 structure, 96-100
Index 477
transcription, relationship with RNA, 101, 296 allosteric, 90-1
DNA polymerase, 292-6 co-operativity, 91
DNA synthesis kinetics, 91
accuracy, 296 structure, 92
viruses, 294--6 coenzyme Q, 45, 48, 164
docosahexaenoic acid, 37, 263-4 coenzymes, 84--90
dolichols, 45 concentration, 76
Donnan equilibrium, 18 diagnostic uses, 79
dormancy, see plant growth and development digestive, inhibitors, 410-11
dwarfing, pot plants, growth retardants, 382 factors affecting rate of enzyme catalysed reactions,
dynein,9 76-90
induction/repression, 289, 304--5
Eadie-Hofstee plot, 78 inhibitors, 81-4
ecdysones, 46 allopurinol, 84
EFAs, see essential fatty acids competitive, 81
EFE, see ethylene-forming enzyme herbicides, 83-4
eicosanoids, 188 insecticides, 82-3
effects, 190 irreversible, 81
structure, 189 non-competitive, 81-2
eicosapentaenoic acid, 37, 263, 264 pharmaceuticals, 84, 85
electron transport chain, mitochondria, 161-71 practical applications, 81-4
AIP yield, 168 sulphonamides, 84
complexes, 164--6 kinetics, 76-81
components, 163-4 mode of action, 72-3
cytochrome b, c1 complex, 165 PABA,84
cytochrome oxidase, 165-6 pH,79-81
NADH-dehydrogenase complex, 164 reaction profile uncatalysed and catalysed reactions,
NADH-ubiquinone oxidoreductase, 164--5 73
organisation in inner mitochondrial membrane, 167 redox coenzymes, biological oxidations, 84--7
shuttle reactions, 169-70 RNA polymerases, 101
succinate dehydrogenase complex, 164--5 temperature, 79
succinate-ubiquinone oxidoreductase, 164--5 EPA, see eicosapentaenoic acid
ubiquinone cytochrome c oxidoreductase, 165 epinasty, see plant growth and development
electron transport coupling, AIP synthesis, 166-8 ergocalciferol, see vitamin D2
electron transport system, chloroplasts, 221-2 ergotism, 55
integration, 225 erucic acid, 37, 38
Z-scheme, 221-2 Escherichia coli
elongation, polyunsaturated fatty acids, metabolic trans- DNA structure, 98
formation, 264 fatty acids
endocrine control, lactation, 454--5 chain length specificity, 260
endonucleases release to ACP, 261
restriction, DNA manipulation, 308 essential amino acid, see amino acids, essential
specificity, 309 essential fatty acids, 263-5
endoplasmic reticulum, 46-7 deficiency, 265
energy storage in animals and plants, 133-4 etacelosil, 389
enzyme-catalytic reactions, NAD+ and NADP+, 84--87 ethanol production, 214--16
enzyme-substrate complex, 72 ethylene
covalent intermediate formation, 74--6 analogues, ethylene-like responses, 389
rate of reaction, 76 biochemistry, 388-9
dependance on substrate concentration, 77 modulation of effects on plants, 389
enzymes, 71-93 physiological activities, 389-92
acid-base catalysis, 74 synthesis and transport, 389
active site, 72, 73 ethylene-forming enzyme, 389
activity eukaryotes
contributory factors, 73--6 defined,3
environment, 74 DNA structure, 98-100
measurement, 78-9 photosynthesis, 217-18
substrate and reaction intermediate stability, 74 promotor sequences, 307
substrate proximity, 73-4 regulation, protein synthesis, 305-6
478 Index
eukaryotic pathway, fatty acid desaturation, 263 malonyl-CoA production, 253-6
extrinsic proteins, see peripheral proteins in mammary gland, 452
NADPH production, 253, 254, 255
FJ',-ATPase, structure, 168 reactions, 257-9
famesol, structure, 47 tissue and subcellular location, 252
famesyl pyrophosphate tissue uptake, lipoprotein lipase action, 403-4
condensation to produce squalene, 275 trans/cis double bonds, 36
synthesis, 274 unsaturated, formation, 262-5
fats and oils uptake from blood, 452, 453
biosynthesis, seed development, 357 world production, 42
deposition in adipocytes, 430-1 fermentation pathways, 207-15
physicochemical properties, 42 herbages, 214
world production, 42 ferredoxin, 222-5, 280, 281
fatty acid elongases, 261-2 ferritin, 130
fatty acid synthetases, type III, 261-2 ferulic acid, 58
fatty acid-binding proteins, 402-3 fishmeal, amino acids, 138
fatty acids, 35-9, 40 flagella and cilia, 4, 8-9
activation and transport to mitchondrial matrix, 175 flavine adenine dinucleotide, 84-7
a-oxidation, 182-3 regeneration by electron transport chain, 162
~-oxidation, 174--82 structure, 109
w-oxidation, 182-3 flavine mononucleotide (FMN), 84-7, 109
branched-chain, 39 flavones, 60-1
structure, 39 flavonoids, 58-60
composition of vegetable and animal lipids, 40 anthocyanins, 58-60
cyclopropane, 39 flavones, 60
defined,35 structure, 61
desaturation flavoproteins, 108, 163-4
animals, 262-3 'flip-flop' transition, lipid bilayer, 314
plants, 263 flower storage life, see plant growth and development
distribution in milk fat, 453-4 flowering, see plant growth and development
elongation, 261-2 FMN, see flourin mononucleotide
essential, 263-5 fodder crops, fermentation, 214
hydroxy and cyclic, 39 folic acid, 114-15
iso- and anteiso-branched, 39 deficiency, 111
long-chain, synthesis, 256-61 forage legumes, 348
mammary gland synthesis, 452-3 formaldehyde, added to silage, 349
melting point, 38 formic acid, added to silage, 349
metabolic regulation, 327-8 free energy, 87-90
mobilization, 190 freezing injury, see plant growth and development
modifications in mammary gland, 453-4 freezing point
monounsaturated, defined, 35-6 depression, 16-18
nomenclature and structure, 36-7 osmotic pressure formula, 17
oxidation, 173-89 fructose, 23
oxidation in plants, 154 phosphorylation, 146
peroxidation, 183-9 structures, 24
prevention, 185 conversion to glucose-6-phosphate, 240
polyunsaturated, 263-4 hydrolysis, 240-2
defined,36 phosphorylation, 143
nomenclature, 36, 37 production,238-40
release from fatty acid synthetase, 261 fructose-2-6-bisphosphate
saturated and unsaturated straight-chain, 35-9 control of glycolisis and gluconeogenesis, 327, 328
synthesis, 251-77 fructose-6-phosphate
acetate production, 209, 210 conversion to fructose-I, 6-bisphosphate, 143
acetyl-CoA production, 252-3 fate, 242-3
acyl carrier protein, structure and function, 256-7 production from glucose-6-phosphate, 143
branched-chain, 260-1 Fruit
chain length specificity, 259-60 development and composition, 353-4
cytoplasmic acetyl-CoA production, 254 function, 10
de novo, 252-61, 452-3 see also plant growth and development
Index 479
fruit ripening, 354--6 glucagon, effects of, 255, 325, 327-8, 431
colour changes, 354 glucocorticoids, growth manipulation, 439
ethylene effects, 355-6, 389-91 glucomannans, 34
respiration, 355-6 gluconeogenesis, 89, 237-43
structures of fruit acids, 354 control, 238, 240-2, 327
texture changes, 354--5 pathway outline, 238
see also plant growth and development from propionate, 243-4
futile cycles starting materials, 237-8
adipocyte fat deposition, 430 glucose, 23
heat production, 418 absorption and utilization in digestion, 398-9, 400
glucose and glucose-6-phosphate, 89 formation via Cori cycle, 208-9
phosphofructokinase and fructose-l,6-bisphosphate, structures, 24, 25
242 glucose permease system, 316, 318
pyruvate and phosphoenolpyruvate, 238, 241 glucose phosphorylation, 88-9, 141-3
triacylglycerol, 419 glucose-6-phosphate
oxidation in pentose phosphate pathway, 201
G-proteins, 321,438,439,440 conversion to glucose, 243
GA, see giberellic acid oxidation in glycolysis, 141-3
galactans, 32 glucosinolates, 32-3
galactolipids, ruminant animals, 404 glucuronic acid, structure, 25
see also glycolipids glutamate (glutamic acid)
galactopoesis, 455 oxidative deamination, 196
galactosan pectins, 31 structure, 25
galactose see also transemination reactions
conversion to glucose-I-phosphate, 146, 147 glutamate synthase (GOGAT), ammonia assimilation,
phosphorylation, 146 282-4
structure, 25 glutamine synthetase (GS), ammonia assimilation, 282-4
galacturonic acid, structure, 25 glutelins, properties, 67
gallic acid, structure, 60 glyceraldehyde, optical isomers, structure, 21
gangliosides, 44, 45, 270 glyceraldehyde-3-phosphate, 142-5, 201-2, 204, 239
gelatin production, 427 glycerides, absorption from small intestine, 402-3
gene cloning, 309-10 glycerol-3-phosphate
polymerase chain reaction, 309, 310 formation from dihydroxyacetonephosphate, 266-7
gene coding, seed development, 357 production from glycerol, 267
genetic code, 297, 299 shuttle, reactions, 170
genetic engineering, 308-11 glycerol-3-phosphate pathway, 266-7
antisense RNA, 310 glycerophospholipids, 42-3
DNA insertion, vectors and methods, 310-11 structure, 43
DNA isolation and synthesis, 308-9 glycogen, 26-7, 29
enzymes in DNA manipulation, 308 metabolism, control by covalent modification, 323-5
gene cloning, 309-10 muscle tissue, 464--5, 468
restriction fragment length polymorphism, 310 synthesis, 246
screening techniques, 310 utilization in glycolysis, 146-7
genetic' fingerprinting', 309 glycogen phosphorylase, 146-7, 323-5
geraniol, structure, 46 glycogen synthetase, 246, 323-5
germination of seeds, 340-42 glycolipids
beer and whisky production, 341-2 breakdown, 192
lipid breakdown, 342 synthesis, 268-70
protein breakdown, 342 glycolysis, 141-7
starch breakdown, 340-1 ATP production, 145, 154
see also plant growth and development control, 325-8
Gerstmann-Straussler syndrome, 69 pathway, 142, 144
GH, see growth hormone from sugars other than glucose, 145-7
gibberellins, 379-82 stage one (glucose to triose phosphates), 142-3
biochemistry, 379 stage two (triose phosphates to pyruvate), 143-5
physiological activities and applications, 379-82 glycosylglycerides, 43-4
synthesis and transport sites, 379 structure, 44
starch regulation, 341 glyoxylate cycle, 156-9, 342
globulins, properties, 67 reactions, 158
480 Index
glyoxysomes,4,7-8 effect of environmental temperature, 413-15
glyoxylate cycle, 156-9 futile cycles, 418
~-oxidation, 179, 181 ion gradients maintenance, 415-16
glyphosate, 290 muscular activity, 418-19
structure, 84 protein turnover, 416-17
glyphosine, plant growth and development, 392 shivering, 418
GMP formation, 292 uncoupled phosphorylation, 171, 419
Golgi bodies, 4, 7 heat-shock proteins, 93, 194, 303-4, 372
role in milk synthesis, 449-50, 452 a-helical chains, 62-3, 64, 65, 70
role in protein synthesis, 302-3 hemiacetals, structure, 22
gondoic acid, 37, 38 hemicellulose, 29, 31, 344
Gram-negative/positive bacteria, 6, 442-4 herbages, 137-8,214,344,347-8
grasses herbicides
carbohydrate content, 138 auxins as, 375-6
composition, 137 enzyme inhibitors, 83-4
hays, 348-9 inhibition of acetoxy acid synthetase, 83, 288, 289-90
preservation, 214 inhibition of EPSP synthetase by glyphosate, 83, 286,
silage, 214, 349 289-90
temperate, 347 inhibitors of amino acid biosynthesis, 289-90
tropical, 348 photosynthesis, 234
greenhouse gases, 370-1 heterogeneous nuclear RNA, 296
groundnuts, amino acids, 138 heterolactate pathway, 214, 215
growth, animals, 421-5 heteropolysaccharides, 26
adipose tissue, 429-31 hexose monophosphate shunt, see pentose phosphate
bone, 427-9 pathway
collagen, 425-7 hexoses,23
general principles, 422 high-temperature stress, see plant growth and develop-
manipulation, 431-45 ment
antibiotics, 440-5 high irradiance response, 370
~-agonists, 435-8 histones, 98, 100, 305--{i
glucocorticoids, 439 HMG-CoA, synthesis, 271-2
growth hormone, 431-3 HMG-CoA reductase, phosphorylation/dephosphoryla-
ionophore/non-ionophores, 440-5 tion mechanism, 272
insulin-like growth factors, 431-3 hnRNA,296
oestrogens and androgens, 433-5 Hogness box, 306
thyroid hormones, 439 homocysteine, conversion to methionine, 117
muscle, 421-5 homolactate pathway, 214
protein accretion, 425 homopolysaccharides, 26
rates and patterns, 422-4 hormone receptors, 91, 374, 387, 432-3, 435, 436, 437-8
regulation and manipulation, 421-45 humans, vitamin deficiencies, 110-12
growth hormone, see also somatotropin, 431-3 hydrochlOric acid secretion, transport mechanisms, 406,
growth inhibition, see plant growth and development 407
growth retardants, see plant growth and development hydrogen bonds, 11-13, 29, 63-4, 97-8
GTP hydrolases, 72
conversion to ATP, 153-4 hydroxylysine, 177, 425-6
formation in TCA cycle, 151-3 hydroxymethylglutaryl-CoA, see HMG-CoA
requirement for protein synthesis, 300, 302 hydroxyproline, presence in collagen, 66, 117,425-6
GTP-binding protein, see G-proteins in cell wall proteins, 344
guanine, 96, 98 hypocalcaemia, parturient, 128
guanosine-5' -monophosphate, see GMP hypoxanthine, 85, 96
guanosine-5' -triphosphate, see GTP
gymnosperms, lignin, 57-8 lAA, see indole-3-acetic acid
lAA oxidase, 374
haemoglobin, structure, 66 IGFs, see insulin-like growth factors
hays, 136,348-9 immunoglobulins, 91-3
addition of propionic acid, 349 indole-3-acetic acid (IAA), 374-5
'head-to-tail' condensations, 274, 379, 381 see also auxins
'head-to-head' condensations, 274 inositol hexaphosphate (phytic acid), cereal seeds, 339-40
heat production, 414-20 insecticides
Index 481
carbamate, 82 role of Colgi body, 449-50
enzyme inhibitors, 82-3 larkspurs, alkaloids, 57
organophosphorus, 82 lasalocid ionophore, 442
insulin, bovine, primary protein structure, 64 lathyrogens, 55
effects of, 255, 328, 329, 430, 431 Lathyrus odoratus (sweet pea), 55
insulin receptor, mode of action, 433 Lathyrus sativus (chickpea), 55
insulin-like growth factors, 431-3 lauric acid, 36--8
integral proteins, 314 leaves, 10
intermediate filaments, 9 development, see plant growth and development
intrinsic proteins, see integral proteins light absorption, 369
intron removal, 297 lectins, action, 34
diagrammatic representation, 298 legumes
iodine, 130-1 forage, 348
ionization, water, 12-14 inhibitors of digestive enzymes, 410
ionophores lathyrogens, 55
examples, 441 seed composition, 336--9
growth manipulation, 440-3 see also nitrogen fixation
structure, 442 Leucaena leucocephala, mimosine, 54-5
iron, 129-30 leukotrienes, 118, 188-9
oxidation, 163 LHC I, 223, 224
plant nutrition, 363 LHC II, 223-5
iron-sulphur proteins, 130, 164, 223-5 ligases,72
isocitrate (isocitric acid), conversion to a-ketoglutarate, light, see plant growth and development
150, 151 light effect mediators, 229
isoelectric point, 68 light harvesting complex see LHC I; LHC II
isomerases, 72 light reactions, photosynthesis
isoprene, structure, 46 cytochrome b-f complex, 225
electron transport system, 221-2
jack bean seeds, canavanine, 52 light absorption, 218-21
juvenile hormones, 46 photosystem I, 222-3, 224
photosystem II, 223-5
ketone bodies plastocyanin, 225
synthesis, 181-2 resonance energy transfer, 221
pathway, 182 lignin, 57
ketosis, adipose tissue and liver relationship, 330, 332 composition, 58
kidney, 10 plant cell walls, 345-6
kinesin,9 digestion, 346
Kraft process, chemical pulping of wood, 350-1 structure of precursors, 59
Krebs cycle, see tricarboxylic acid cycle wood,350-1
lignin-free radicals, polymerization, 346
laburnum, alkaloids, 55--7 lignoceric acid, 37
lactate (lactic acid) limonene, structure, 46
involvement in propionate production, 209 Lineweaver-Burk plot, 78
muscle, 214, 465, 468 linoleic acid, 36, 37, 38, 263-5
production, 207--8 linolenic acid, 37, 38, 263-5
silage, 214-5 lipase, see triacylglycerollipase; pancreatic lipase; phos-
use in Cori cycle, 208-9 pholipase
lactation lipid bilayer, 313-14
dairy cow, lactation curve, 448 structure, 315
endocrine control, 454-5 lipids
ketosis, 330 absorption from small intestine, 402-3
manipulation, 455--7 cholesterol, 403
dietary, 456--7 chylomicron formation, 403
exogenous hormones, 457 glycerides, 402-3
origin of components of milk, 448-54 phospholipids, 403
see also mammary gland uptake by body tissue, 403-4
lactogenesis, 454, 455 animal, fatty acid composition, 40
lactose, structure, 26 breakdown, 189-92
lactose synthesis, 243-6 fatty acid oxidation, 173--83
482 Index
a-oxidation, 182-3 plant nutrition, 363
il-oxidation, 174-82 manioc, 352
w-oxidation, 183 mannans,34
deposition of fat in adipose tissue, 430-1 mannose, structure, 25
digestion meat, see carcass; skeletal muscle
monogastric animals, 399-404 membrane signal transduction, 321-2
poultry, 406 GTP-binding protein, 321
ruminant animals, 404-6 membrane lipids, 313-15
in membranes, 313 tocopherol, 186
chemical analysis, 313-14 membrane proteins, classification, 314-15
peroxidation of fatty acids, 183-9 membrane structure, fluid mosaic model, 314-15
physicochemical properties, 42 membrane transport, 315-22
seeds, 337 co-transport, 316
synthesis, 251-77 bicarbonate transport by erythrocytes, 316-17, 319
fatty acids, 252-65 protonllactose symport, 319-20, 321
glycolipids, 268-70 glucose permease system, 316, 318
phospholipids, 268 group translocation, 320-1
sphingolipids, 270-1 protein channels, 316, 317
terpenes and sterols, 271-7 uniport systems, 316
triacylglycerols, 265-8 menaquinone, see vitamin K
temperate grasses, 347 menthol, structure, 46
vegetable, fatty acid composition, 40 messenger RNA (mRNA), 101, 296-311
lipoxygenase, 188-9 metabolic body weight, 414, 423
liver, 10 metabolic regulation
lodging, cereals, growth retardents, see plant growth and allosteric regulation, 323
development coordination in different tissues, 328-32
Lupinus spp, alkaloids, 55--7 covalent modification, 323-5
lyases,72 pathway coordination, 325--8
lysergic acid, structure, 56 principles, 322-3
lysosomes, 4, 7 methane production, 213, 443
methionine
magnesium, 128-9 content in plants, 136, 138, 338-9
magnesium-ATP complex, structure, 129 precursor of ethylene, 388-90
plant nutrition, 362 role in protein synthesis, 299-300
Maillard reaction, 352 synthesis, 177, 285, 360
malate, conversion to oxaloacetate, 151-3 methylmalonyl-CoA
malate-aspartate shuttle, reactions, 169-70 conversion to succinyl-CoA, 117
maleic hydrazide, plant growth and development, 392 excretion in vitamin B12 deficiency, 111
malate (malic acid), structure, 354 in branched-chain fatty acid synthesis, 260
malignant hypothermia, 468-9 mevalonic acid
malonyl-CoA, synthesis of long-chain saturated fatty conversion to
acids, 256-61 isopentenyl pyrophosphate and dimethylallyl
malonyl-CoA production, 253-6 pyrophosphate, 273
animals, 254-5 squalene, 273-7
bacteria, 256 synthesis, 271-2
plants, 256 Michaelis-Menten equation, 76-7, 90
maltose, structure, 26 micronutrients, trace elements, 362
mammary gland, 10 milk,134
amino acid supply, 451 colligative properties, 18
energy supply, 454 composition, 136
fatty acids milk components, origins, 448-9
modifications, 453-4 milk fats, 452-4
synthesis de novo, 452-3 distribution, 453-4
uptake from blood, 453 milk fever, 128
goat, alveoli, 449 milk pasteurization, alkaline phosphatase, 79
see also lactation milk proteins, 451-2
manganese origin, 451
functions in animals, 131 quantities in cow's milk, 452
in glycolysis, 142 synthesis, 451-2
Index 483
mimosine, 54-5 nicotinamide, structure, 113
minerals, 127-31 nicotinamide adenine dinucleotide, see NAD/NADH
carcass composition, 134--5 nicotinamide adenine dinucleotide phosphate, see
in plant nutrition, 354-63 NADP/NADPH
levels in plant tissues, 360 nicotine, structure, 56
seeds, 339-40 nicotinic acid, 108-9, 113
toxic effects in plants, 362-3 deficiency, 110
mitochondria, 4, 8 Niemann-Pick disease, 271
coupled, 169 night blindness, 121
electron transport chain organization, 167 nightshades, alkaloids, 57
glyoxylate cycle, 157, 159 nitrate assimilation, 279-81
matrix function, 4 nitrite reductase, 279-81
photorespiration, 229-31 nitorgen metabolism
protein synthesis, 306-7 plants, 193-7, 199,279-84
structure, 8, 163 plant nutrition, 364--6
mitochondrial membrane, inner, 4 nitrogen compounds, transport in plants, 364--5
molecular recognition, 91-3 nitrogen fixation, 281-2
molybdenum molecular biology, 282
animal nutrition, 131 nitrogen metabolism, animals, 193-9
nitrate reductase, 280 ammonia excretion, 197
plant nutrition, 363 urea cycle, 197-9
Monensin, ionophore, 441-3 nitrogenase, action, 281-2
2-monoacylglycerol pathway, 265-6 non-esterified fatty acids (NEFAs), 174,453
monocots non-protein nitrogen, see NPN
hemicelluloses, 29 non-ruminants, see monogastric animals
lignin, 57 noradrenaline, structure, 437
monogalactosyldiacylglycerols, 44, 268-70 Northern blotting, 310
monogastric animals NPN (non-protein nitrogen), 337, 339
digestion and absorption of nutrients, 395-411 seeds, 337-8
monoglycosyl ceramides, 270 rumen microbial protein synthesis, 199, 409-10
morphactins, plant growth and development, 392 nucleic acids
morphine, structure, 56 structure, 95-104
mRNA, 101, 296-311 synthesis, 291-297
muscle, see skeletal muscle see also DNA; RNA
mutagenesis, site-specific, 311 nucleoid,6
myofibril structure, 459--61 nucleolus, 6
degradation by enzymes, 466 nucleosides, 95, 96
myoglobin, 465 nucleosomes, 99-100
muscle tissue after death, 466-7 structure, 100
myosin II, 459--61 nucleotides, 95, 96
myristic acid, 36, 37 synthesis, 292-4
nucleus, 4, 6
N-acetyl-glucosamine, structure, 25 nuclear envelope, 6
Na-K-ATPase, 317-20, 415-16 nutrition
NAD/NADH, redox coenzymes, 84--7 antinutrients
see also glycolysis; tricarboxylic acid cycle; electron alkaloids, 55
transport chain cyanogenic glucosides, 33-4
NADH, transport from cytoplasm to mitochondria, 169 glucosinolates, 32-3
NADH--<lehydrogenase complex, electron transport lathyrogens,55
chain, 164 lectins,34
NADH-ubiquinone oxidoreductase, electron transport non-protein amino acids, 52-55
chain, 164 phenolics, 57--60
NADP/NADPH, redox coenzymes, 84--7 phytic acid, 339-40
see also pentose phosphate pathway; fatty acid synthe- protease inhibitors, 79, 410-11
sis; photosynthesis; cholesterol synthesis saponins, 33
NEFAs, see non-esterified fatty acids SMCO,55
nervonic acid, 37 plants, 359--66
niacin, see nicotinic acid nutritional role in animals of
nickel, plant nutrition, 363 amino acids, 287-9
484 Index
essential fatty acids, 263-5 pellagra, 108-9, 110
minerals, 127-31 pentose phosphate pathway, 201-6, 253-5, 280, 454
vitamins, 105-27 alternative modes of operation, 205
oestrogens and androgens oxidative reactions and NADPH production, 201, 203
growth manipulation, 433-5 rearrangement reactions, 201-5
mode of action, 434-5, 436 regulation, 206
natural, 433-4 pentoses, 21-3
synthetic, 434 peptide bonds, 60-1
treatment responses, 434 hydrogen bond formation, 64-5
oilseed rape, 32, 40, 42, 336--7 structure, 61
Okazaki fragments, 292 peripheral proteins, 314
oleic acid, 37, 38, 262-4 peroxidation
oxidation, 179 effects in living organisms, 186--8
oligonucleotide-directed mutagenesis, see mutagenesis, fatty acids, 183-9
site-specific prevention, 185
w-oxidation, 183 measurement, 185-6
operons, 304-5 peroxisomes, 4, 7
opsin, 121 l3-oxidation, 179-81
optical isomers, 20, 21 photorespiration, 229-31
organelles, 4-9 PFK, see phosphofructokinase
DNA,100 PFK2, see phosphofructokinase 2
protein synthesis, 306--7 pH
organophosphorus insecticides, 82 chloroplast, 225
organs, functions, 10 digestive system, 400, 405, 406
orthophosphate cleavage of AlP, 90 effect on proteins, 68
osmolarity, 17 meat, 214, 468-9
osmotic pressure, 16--17 enzyme function, 79-81
biological fluids, 18 buffering in biological systems, 12-15
ideal solution, formula, 17 silage, 214
osteolathyrism, symptoms, 55 water, 12-14
osteomalacia, calcium deficiency, 128 pharmaceuticals, inhibitors of enzymes, 84, 85
osteoporosis, calcium deficiency, 128 phaseic acid, 387-8
oxaloacetate, 150-3 phenolics, 57-60
oxidative phosphorylation, 161-71 flavonoids, 58-60
regulation, 169-71 lignin, 57
oxidoreductases, 72 structure, 58, 59,60
oxygen levels and plant growth, see plant growth and tannins, 57-8
development phenylalanine ammonia lyase (PAL)
oxythiamin, structure, 107 activation, 284-5
catalytic reaction, 287
PABA (p-aminobenzoic acid), 84, 85 phosphate esters, 128, 361-2
paclobutrazol, 382 hydrolytic free energy, 87-9
PAL, see phenylalanine ammonia lyase phosphatidic acid, 267-9
pale, soft, exudative pork, 469 phosphocreatine, muscle tissue, 464-5
palmitic acid, 36, 37, 38 phosphoenol pyruvate, structure, 84, 144
oxidation, AlP yield, 176--8 phosphofructokinase (PFK), 142, 143
see also fatty acids control,240-2
palmitoleic acid, 37, 38, 264 phosphofructokinase 2 (PFK2), 327-8
pancreatic lipase, small intestine, 400-2 phosphogluconate pathway, see pentose phosphate path-
pantothenic acid, 112-13 way
deficiency, 110 3-phosphoglycerate
papain, 74 structure, 144
paper making, 350-1 see also glycolysis; starch synthesis; Calvin cycle
parturient hypocalcaemia, see hypocalcaemia, parturient phospholipases, sites of action, 191-2
PCR, see polymerase chain reaction phospholipids, 42-3, 268-9
PDH, see pyruvate dehydrogenase breakdown, 191-2
pectin~32,344-5,354-5 digestion and absorption in non-ruminants, 403
structure, 31 digestion and absorption in ruminants, 404
pectin methyl esterase, 391 membranes, 313-15
Index 485
synthesis, 268-9 cambium growth and differentiation
phosphorus, 128 auxins, 378
plant nutrition, 361-2 gibberellins, 382
phosphorylation carbon dioxide levels, 370-1
oxidative, 161-71 cell division
uncoupled, heat production, 171, 419 cytokinins, 384-6
phosphotransferase system, 320-1, 322 chilling injury, 371-2
photorespiration, 229-32 cold requirement for growth
factors affecting rate, 230-2 gibberellins, 379-80
pathway, 231 correlative effects
photosynthesis, 217-35 growth retardants, 382-3
C4 plants, 232-3 cytokinins, 384-7
diagrammatic representation, 233 dormancy
carbon dioxide levels, effect of, 372 bud, abscisic acid, 388
chloroplasts, 4, 8, 218-19 seed, abscisic acid, 388
control, 229 seed, gibberellins, 382
dark reactions (Calvin cycle), 226--9 epinasty
herbicides, 234 ethylene, 391
light intensity, effect of, 368 ethylene, 388-92
light reactions, 218-25; see also light reactions flower storage life
pigments and light absorption, 218-21 ethylene, 392
rates in different types of plant, 234 flowering, 353
Z-scheme, 221-2 auxins, 378
photo system I and II, 222-5 ethylene, 392
phototropism, see plant growth and development gibberellins, 380
phytic acid (inositol hexaphosphate) freezing injury, 371
cereal seeds, 339-40 fruit development
structure, 340 auxins, 378-9
phytochrome-mediated responses, see plant growth and gibberellins, 382
development, light growth retardants, 383-4
phytol, structure, 47 fruit ripening
pigs ethylene, 389-91
pale, soft, exudative pork, 469 germination
vitamin deficiencies, 110-12 gibberellins, 382
a-pinene, structure, 46 gibberellins, 379-82
plant cell walls, 6, 29-32, 344-7 growth inhibition
arabinogalactans, 32, 344 abscisic acid, 388
cellulose, 29-30 growth retardants, 382-4
component linkage, 344-5 high-temperature stress, 372-3
control of cell expansion, 346--7 hormones, see named hormones
hemicellulose, 29, 31, 344-5 leaf development
lignin, 57-8,345-6 cytokinins, 386--7
pectin,31-2,344-5,354-5 epinasty, ethylene, 391
xyloglucan, 29-31, 344-5 light
plant growth and development high-irradiance responses, 370
abscisic acid (ABA), 387-8 photosynthesis effects, 368
abscission phytochrome-mediated responses, 368-70
fruit, ethylene, 391 lodging, cereals
leaf, ethylene, 391 growth retardants, 382
apical dominance oxygen levels, 370-1
auxins, 378 phototropism, 370
cytokinins, 387 auxins, 377
assimilate movement pot plants
abscisic acid, 388 growth retardants, 382
atmosphere responses, 370-1 regulation, 367-92
auxins, 374-9 reproductive growth, 353-7
callus differentiation root development
auxins, 378 auxins, 378
cytokinins, 384-6 salt stress, 373-4
486 Index
senescence, 366 structure, 27-32
cytokinUns, 386-7 polyunsaturated fatty acids, 36, 263--5
flower, ethylene, 392 metabolic transformation by desaturation and elonga-
stem elongation tion, 264
auxins, 377 precursor fatty acids, 264
ethylene, 391 porcine stress syndrome (PSS), 468
stomatal opening pork, pale, soft, exudative, 469
abscisic acid, 387-8 pot plants, dwarfing, growth retardants, see plant growth
stress responses, 371-4 and development
abscisic acid, 387-8 potassium, 129
temperature stress, 371-3 concentration in mammalian cell, 129
vegetative growth, 343--52 plant nutrition, 362
gibberellins, 379 stomatal opening, 387
vernalization, 370 potatoes, 351-2
water stress, 373 alkaloids, 57
abscisic acid, 387 poultry
plant materials lipid digestion, 406
composition and nutritional use, 137-8, 336 vitamin deficiencies, 110-12
water content, 136 pre-slaughter stress (PSS), catecholamine hormones, 468
plant nutrition, 359--66 Pribnow box, 303
biochemical functions of nutrients, 359--62 primary electron acceptor, photosynthesis, 221
boron, 363 prion proteUns, 69-70
calcium, 362 multiplication, 70
carbohydrate energy store, 133-4 prokaryotes
carbon assimilation, 217-35, 364 cells, 3
chlorine, 223, 363 RNA polymerase binding sites on DNA, 304
coppe~225,363,389 DNA structure, 9S-100
iron, 223--5, 363 fatty acid desaturation pathway, 263
magnesium, 362 photosynthesis, 218
manganese, 223,363 regulation, protein synthesis, 303--6
molybdenum, 280, 363 prolamins, 67, 337-9
nickel,363 proline, hydroxylation Un proteins, 66, 425--6
nitrogen,279-84,359,364-6 promotor sites and sequences, 303, 306
phosphoru~361-2 propionate
potassium, 362 conversion to glucose, 155, 243
sulphur, 359--61 formation
zinc, 363 digestive systems, 209
see also photosynthesis glucose fermentation, 211
plasma glucose, 142-3, 329 propionic acid, added to hays, 349
energy source for muscle, 464 prostacyclin, structure, 189
plasma lipids, 403--6, 435 prostaglandin, structure, 189
energy source for muscle, 464 prostaglandin analogues, 18S-9
plasma membrane, 3-4 protein breakdown, 193--9
plasmids, vectors for DNA clonhlg, 310-11 germination, 342
plastocyanUn, 224, 225 protein half-life, 417-18
plastoquUnone, 45, 48, 224-5 senescence, 366
J3-pleated sheet, protein structures, 63, 64, 65, 70 protein channels, membranes, 317
PMF, see proton motive force protein digestion
polygalacturonans, 32 monogastric animals, 406-9
polygalacturonase, 391 ruminants, 409-10
polyglycosylated ceramides, 270 protein metabolism, in rumen, summary, 410
polymerase chain reaction (peR), gene cloning, 309, 310 protein synthesis, 297-308
polymerase/depolymerase activity of DNA polymerase, amino acid activation, 297-9
296 chloroplasts, 306-8
polyprenols, 45 elongation, 300
polysaccharides, 26-32 eukaryote regulation, 305--6, 425, 426
cellulose synthesis, 248-50 initiation,299-300,301
glycogen synthesis, 246 initiation factors, 300, 426
starch synthesis, 246-8 location, 301-3
Index 487
mitochondria, 306--8 pyrimidines, 95-104
orgenelles, 307 bases, 96
post-translational modification, 300-1 biosynthesis, synthesis, 292
prokaryote regulation, 303-5 pathway, 294
prokaryotes, 302 defined, 95--6
regulation, 302--6, 425, 426 nucleosideslnucleotides, 96
seed development, 357 pyrithiamin, structure, 107
termination, 300 pyrophosphate cleavage of ATP, 90
protein turnover, 194, 366, 417-18 pyruvate (pyruvic acid), 141
heat production, 416-17 C. photosynthesis, 232-3
proteins, 61-70 control of production and use, 238
a-helix,63,64,69,70 conversion to acetyl-CoA, 150
f3-pleated sheet, 63, 64, 65, 70 conversion to lactate, 161-2
carbohydrate addition, 66, 301, 302-3 metabolism via TCA cycle, 149-53
cereal, 337-9 pyruvate dehydrogenase (PDH), 150
denaturation, 68-9 pyruvate formation, 144, 145
function and structure, 61--6
hea t effect, 69 quinolizidine nucleus, structure, 56
hydroxylysine, 177, 425--6 quercitin, 108
hydroxyproline
in cell wall, 344 ractopamine, structure, 437
presence in collagen, 66, 117, 425--6 ragwort, alkaloids, 55
ionic strength, 67--8 rapeseed, see oilseed rape
legumes, 339 receptors, 91, 374, 389, 431-3, 435, 436, 437--8
organizational levels, 62 redox coenzymes
pH effect, 68 biological oxidations, 84-7
plants and animals, as nutrients, 135--6 structure, 85, 86
primary structure, 62, 63 resonance energy transfer, photosynthesis (RET), 220, 221
properties, 66-9 respiration, 371
quaternary structure, 62, 64--6 fruit ripening, 355--6
salting in/out, 67--8 photorespiration, 229-32
secondary structure, 62-3 restriction endonucleases, 308-9
seeds, 337-9 restriction fragment length polymorphism (RFLP), 310
solubility classification, 67 RET, see resonance energy transfer
specificion~67--8 retinal, structure, 120
temperate grasses, 347 retinaldehyde, structure, 120
tertiary structure, 62, 63-4 retinoic acid, structure, 120
proton motive force (PMF), 167--8, 171,320 retinol, structure, 48, 120
PrP, see prion proteins retronecine, structure, 56
prunasin, 33 RFLP, see restriction fragment length polymorphism
PSE pork, see pale, soft, exudative pork rhamnogalacturonans, 32
PSS, see porcine stress syndrome pectins, structure, 31
PTS, see phophotransferase system rhodopsin, 121
PUFAs, see fatty acids; polyunsaturated fatty acids riboflavin, 108
purines, 95-104 deficiency, 110
bases, 96 ribonucleic acid, see RNA
defined,95--6 ribose, 21, 22
nucleosides, nucleotides, 96 ribosomal RNA (rRNA), 101-2
synthesis, 292 composition of 80S and 70S ribosomes, 104
pathway, 293 ribosome~4,6, 102, 104
pyridoxal, 112-14 ribulose-1,5-bisphosphate (RUBP), 226--8
structure, 113 ribulose-1,5-bisphosphate carboxylase/oxygenase, see
see also vitamin B. Rubisco
pyridoxamine, 112-14 ricinoleic acid, 39
structure, 113 rickets, 112, 124
see also vitamin B. calcium deficiency, 128
pyridoxine, 112-14 RNA,l00-4
structure, 113 antisense, 310, 391
see also vitamin B. mRNA, see messenger RNA
488 Index
polymerases, 101, 296 supplementation in vitamin E deficiencies, 126
relationship with DNA, 101 semen, colligative properties, 18
rRNA, see ribosomal RNA semipermeable membranes, 17-18
splicing, 297--8 Senecio jacobea (ragwort), alkaloids, 55
structure, 100 senescence, see plant growth and development
synthesis, 101, 296--8 sheep
translation to proteins, 297-300 ketosis, 330
tRNA, see transfer RNA vitamin deficiencies, 110-12
RNA polymerase, 101, 296 shikimic acid pathway, 83, 284-6
binding sites, prokaryotic DNA, 304 shivering, heat production, 418
transcripts, 297 shoots, composition, 344--7
root crops, 137, 351 sialic acid, 33, 44, 271
root development, see plant growth and development silage, 214, 349
roots, 10 addition of formaldehyde, 349
rRNA,101-2 addition of formic acid, 349
composition of 80S and 70S ribosomes, 104 silk,63
Rubisco, 226--32, 307 sitosterol, structure, 49
reactions, 228 skeletal growth, 427-9
synthesis, potassium, 362 skeletal muscle, 10
content in leaves, 347, 366 carcass, 459-69
RUBP carboxylase/oxygenase, see Rubisco changes after death, 465-9
rumen fermentation, 208-10, 211, 212-13, 398, 404, 409-10 cold shortening, 467
effect on lactation, 456--7 conditioning, 467
ruminants myoglobin role, 466--7
absorption, 395-411 stress pre-slaughter effects, 467-9
amino acid supply for protein synthesis, 289 contraction
branched-chain fatty acid synthesis, 260 biochemistry, 459-65
calf, body composition, 135 control, 462-3
digestion contraction cycle, 463
carbohydrate, 398 different types, 464-5
lipid, 404-6 energy provision, 464-5
protein, 409-10 glycogen, 464
digestive compartment sizes, 397 growth, 424-5
ketosis, 330, 332 heat prodUction, 418-19
origin of acetyl-CoA for fatty acid synthesis, 253, 255 mechanism of action, 461-2
vitamin deficiencies, 110-12 metabolism, 208
volatile fatty acids in rumen fluid, 399 phosphocreatine, 464
plasma glucose, 464
S-methyl cysteine sulphoxide (SMCO), 55 plasma lipids, 464
salt stress, see plant growth and development structure, 459-62
saponins, 32 SMCO, see S-methyl cysteine sulphoxide
scrapie, 69-70 smooth endoplasmic reticulum, 4, 6--7
secretory cells, mammary gland, 448-50 sodium, 129
seeds, 10 concentration in mammalian cell, 129
carbohydrates, 336--7 soils, anaerobic environments, 210-11
composition, 336 solanine, solanidine, 56, 57
development, 356--7 somatotropin, 431-3
fat biosynthesis, 357 sorghum, 58, 337-8
protein synthesis, 357 Southern blotting, 310
starch biosynthesis, 356--7 soyabean, 79,188,335,336,339
dormancy, see plant growth and development oil,40, 42, 337
food and agricultural commodities, 335-6 sphingolipids, 44-5
germination, 340-42 structure, 44
lipids, 337 synthesis, 270-1
minerals, 339-40 sphingomyelins, 44--5, 270
non-protein nitrogen, 337, 338 squalene
proteins, 337-9 formation from mevalonic acid, 273-6
selenium, 131 cyclization to sterols, 276--7
in amino acids, toxicity, 52-4 production, 271-6
Index 489
structure, 47 sweet pea, lathyrogens, 55
ST, see somatotropin sweet potato, 352
starch, 26--8 symport transport systems, 316
starch synthesis, 246-8, 356--7
stearic acid, 37, 38 tannins, 57-8
see also fatty acids and proteins, 68
stem elongation, see plant growth and development sorghum, structure, 60
sterculic acid, structure, 39, 41 tartrate (tartaric acid), structure, 354
steroid hormones TATA Boxes, 303, 306
mode of action, 434-5, 436 Tay-Sachs disease, 271
types, 433--4 TBA, see trenbolone acetate
steroids, 45-7 TCA cycle, see tricarboxylic acid cycle
sterols temperate grasses, 347
biosynthesis, 271-7 carbohydrates, 347
cyclization from squalene, 276-7 lipids, 347
structures, 49 proteins, 347
stigmasterol, structure, 49 temperature stress, see plant growth and development
stomatal opening, see plant growth and development terpenes, 45-7, 48, 49
straws, 136--7, 348 biosynthesis, 271-7
stress structures, 46, 47, 48, 49
pre-slaughter effects, 467-9 tetcyclasis, 382
catecholamine hormones, 468 tetrahydrofolic acid (THF), 114-15
substrates, plants, see plant growth and development tetroses, 20-1
enzyme, 72-79 thermogenin, 117, 419
succinate (succinic acid), conversion to fumarate, 153 THF, see tetrahydrofolic acid
succinate dehydrogenase complex, electron transport thiamin, 106-8
chain, 164-5 deficiency, 110
SUCcinate-ubiquinone oxidoreductase, electron transport thiamin pyrophosphate (TPP), structure, 107
chain, 164-5 thick/thin filaments, muscle structure, 461
succinyl-CoA, conversion to succinate, 153 thromboxanes, 188-9
sucrose, 25-6, 348, 351-2, 355, 357 thymine, 96, 98
structure, 26 thyroid hormones, 439
synthesis, 245-6 tissues, functions, 10
pathway, 247 tocopherol, 124-6, 185
sugar acetals, formation, 24-6 content in fats and oils, 185
sugars location in membrane lipid bilayer, 186
five- and six-membered rings, 22-3 structure, 48, 125
hexoses,23 tocotrienol, 124-6, 185
nomenclature, 20 content in fats and oils, 185
optical isomers, 20, 21 structure, 125
pentoses,21-3 tomatoes, ripening, 354, 390-1
reducing and non-reducing, 24 TPP, see thiamin pyrophosphate
ring formation, 22-3 trace elements, 127, 130-1
structure, 20 plant nutrition, 362-3
tetroses, 20-21 transamination reactions, 78, 194-5, 284
trioses, structure, 21 transfer RNA, 101-2
sulphate assimilation, conversion into organic com- clover-leaf structure, 103
pounds, 360-1 transferases, 72
sulphonamides transferrin, 130
enzyme inhibitors, 84 trenbolone acetate, 434-5
structure, 85 triacylglycerollipase, activation via l3-adrenergic recep-
sulphonylureas, herbicides, 290 tor, 190-1,430-1,439
sulphur, 129-30 triacylglycerols, 39-42
plant nutrition, 359-61 2-monoacylglycerol pathway, 265-6
sulphur compounds, volatile, 360 breakdown, 190, 191
sunflower, 339 control, 430, 439
oil, 40, 42 micelle structure, 402
sunshine vitamin, see vitamin D breakdown in small intestine, 400-2
Svedberg units, 102 digestion in ruminant animals, 404-6
490 Index
futile cycle, 419 coenzyme forms, 106
glycerol-3-phosphate pathway, 266-7 deficiencies, 110-12
metabolism, adipose tissue, 430-1 fat-soluble, 106, 119-27
structure, 41 functions, 106
synthesis water soluble, 106-119
animals, 265-7 vitamin A, 119-21
plants, 267-8 deficiency, 112, 121
tricarboxylic acid cycle, 149-59 vitamin BI , see thiamin
ATP production, 154 vitamin BZ' see riboflavin
intermediates replenishment, 155-6 vitamin By see nicotinic acid
links with other metabolic pathways, 153-5 vitamin B6' see pyridoxal; pyridoxamine; pyridoxine
pathway, 152 vitamin BIZ' 115-17
proprionate conversion to glucose, 155 deficiency, 111
regulation, 155-6 vitamin C, 117-18
trioses,21 deficiency, 111
tRNA,101-2 vitamin D, 121-4
clover-leaf structure, 103 deficiency, 112
tropical grasses, 348 vitamin E, 124-6
tubers, functions, 10 deficiency, 112, 126
tuber crops, 351-2 selenium supplementation, 126
vitamin K (menaquinone), 125-7
ubiquinone,45,164-5 deficiency, 112
structure, 48 volatile fatty acids (VFAs), molar proportions in sheep
ubiqUinone cytochrome c oxidoreductase, electron trans- rumen fluid, 399
port chain, 165 synthesis, 2O~1O, 398
ubiquitin, 194,417
uncoupling, electron transport, 169-71, 282, 419 warfarin, 127
uncoupling protein, 419 structure, 125
uniport transport systems, 316 water
unsaturated fatty acids, formation, 262--5 chemical structure, 11-14
uracil,96,100-1 colligative properties of solutions, 15-18
urea ionization, 12--14
fate in ruminants, 199,409-10 water content of plant and animal materials, 134-6
structure, 197 water splitting, photosynthesis, 223-5
urea cycle, 197-9 water stress, see plant growth and development
urease, 79 waxes, 47
uric acid, structure, 197 weight gain, composition, 423
uropygial gland, branched-chain fatty acid production, 260 Western blotting, 310
whisky and beer production, seed germination, 341-2
vacuoles, plant, 4, 7-8 wood, 349-50
vectors, 310-11 paper making, 350-1
vegetable oils Kraft process, 350-1
composition, 40
world production, 42 xanthophylls, 45, 219
vegetative growth of plants, 343-52 xylan,29
vernalization, see plant growth and development xyloglucan, 29-31, 344-5
VFA, see volatile fatty acids xylose, 21-2
viruses
DNA synthesis, 294-6 yeast, addition to feed, 54, 456
RNA synthesis, 296
vectors, 310-11 zinc
vision, vitamin A, 121 animal nutrition, 131
visual cycle, 122 plant nutrition, 363
vitamin(s),105-27 zwitterions, structure, 52

An Introduction To Agricultural Biochemistry

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An Introduction To Agricultural Biochemistry

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AN INTRODUCTION

CHAPMAN & HALL

Chapman & Hall, 2-6 Boundary Row, London SEI 8HN, UK

Chapman & Hall GmbH, Pappelallee 3, 69469 Wcinheim, Gennany

First edition 1998

© 1998 Chapman & Hall

Typeset in 10/12 Palatino by Saxon Graphics Ltd, Derby

ISBN-13: 978-0-412-64390-3 e-ISBN-13: 978-94-009-1441-4

Library of Congress Catalog Card Number: 97-68949

i§ Printed on pennanent acid-free text paper, manufactured in accordance with

List of abbreviations xix

PART ONE: THE CELL AND CELLULAR CONSTITUENTS 1

1 Cell structure and function 3

2 Water and solutions 11

PART TWO: METABOLISM 139

PART THREE: STRATEGIES FOR PROCESSING OF NUTRIENTS IN PLANTS 333

PART FOUR: STRATEGIES FOR PROCESSING OF NUTRIENTS IN ANIMALS 393

2,4,5-T (2,4,5-trichlorophenoxy)acetic acid DNA deoxyribonucleic acid

THE CELL AND CELLULAR CONSTITUENTS

1.1 INTRODUCTION nuclear envelope, are defined as eukaryotic.

Cell structure Function/pathway

Cell membrane Transport of nutrients; hormone/receptor interactions; cell recogni-

Cell wall iii

1.2.8 MITOCHONDRIA 1.2.9 CHLOROPLASTS

Table 1.2 Examples of tissue and organ specialization

2.1 INTRODUCTION Water is a compound of hydrogen and

. H (negative charge) '(G\ Hydrogen ion

Acid Base 2.4.1 STABILIZATION OF PH BY BUFFERS

extremely important in the digestion of plant

CH3COOH ~ CH 3COO- + H+ (2)

Acid Formula Dissociation constant pKa

Formic acid HCOOH 1.77 x 10-4 3.75

/ tems, body fluids exert more or less the same

Fluid Depression of freezing Osmotic pressure

Blood -0.54 302

also means that the total electrical charges are

3.1 INTRODUCTION simple sugars were all found to have the

G alactu ron ic Glucuronic

Cyanogenic glucosides Normally the glucosides are found in the

4.1 INTRODUCTION erties are determined by the type of fatty acids

Trivial name Systematic name Shorthand notation Structure

Monounsaturated fatty acids

Polyunsaturated fatty acids

Trivial name Shorthand notation Melting point (OC) *

Palmitic acid C16:0 60.7

*From Gurr, M.l. and Harwood, J.L. (1991) Lipid Biochemistry, An

Iso-branched fatty acids Anteiso-branched fatty acids

Fat/oil Annual Melting S/U Saponification Iodine value

Beef tallow } 40-50 0.85 190-200 32-47

HO Figure 4.7 The outline structure of sphingolipids.

(b) (c) (d)

(e) (f) (9)

face lipopolysaccharides, peptidoglycans and incorporation into macromolecules such as gly-

Figure 4.9 The structures of farnesol, phytol and squalene.

Vitamin ~ (Menaquinone) Ubiquinone

5.1 INTRODUCTION genetic information carried in the DNA is able

Acidic Imino acids

S-methyl cysteine sulphoxide l3-cyanoalanine

disease' or 'blind staggers'. These amino acids 5.3.3 MIMOSINE

cinnamic acid p-coumaric acid ferulic acid

Table 5.1 Percentage composition of lignin from different sources

Source Coniferyl alcohol Sinapyl alcohol p-Hydroxycinnamyl

CH 20H CH 20H CH 20H