BIOCHEMISTRY

M. ROY MACAULAY

BIOLOGICALLY IMPORTANT MOLECULES

1.1 INTRODUCTION

Living organisms are made up of a limited number of types of atoms, including carbon, hydrogen, oxygen, nitrogen,
sulphur and phosphorus, and ions such as Na +, Mg2+, Cl-, K+, and Ca2+. [Are these all, or can you name some more?]
These elements combine to form molecules which are the building blocks of life. The molecules formed vary in size
from simple molecules such as carbon dioxide and water, to macromolecules such as proteins and nucleic acids.
The small molecules are soluble, easily transported, and frequently enter into the general chemical activities of cells
(metabolism). The larger molecules tend to be used for structural or storage purposes, and some can also be
described as informational molecules, which are concerned with carrying genetic information, and the expression
of that information.

1.2 WATER
1.2.1 Introduction
Water is the most abundant of the small molecules. It makes up between 60-95% of the fresh weight of all
cells/organisms. Evolutionists believe that life evolved in water, because without water, life, as we know it, would
not exist on this planet.
Water is important for a number of reasons:
 It is a vital constituent of living cells
 It forms part of the extra-cellular fluid of multicellular organisms, or as an environment for unicellular
organisms
 It is the medium in which majority of metabolic reactions take place
 It is a reactant in some metabolic reactions
 It transports substances into and out of cells
 It is a solvent for a wide variety of chemicals found in cells, including wastes.
Water has a unique combination of properties that make it essential to the continuance of life. It is a dipolar
molecule, and the molecules are bonded to each other by hydrogen bonds. [The structure of water] Hydrogen
bonds are weaker than covalent bonds, but in totality, they cause the water molecules to cling to each other.
Without hydrogen bonds, water would boil at -80°C and freeze at -100°C, making life impossible. However, because

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of hydrogen bonds, water boils at 100°C and freezes at 0°C, making it a liquid at temperatures that are suitable for
life.
1.2.2 The biological significance of water
1. Solvent properties: Water is an excellent solvent for polar substances. This is because water has a high
dielectric constant, that is, it resists the separation of its charges when placed in an electric field (or when an
electric current is passed through it). Rather, water causes the separation of polar substances into their
constituent ions. On contact with water, the polar groups or ions are surrounded by water molecules which
separate the ions or groups from each other, causing them to behave almost independently.
This property of water makes possible the majority of the cell’s reactions which take place in aqueous solutions.
It also enables water to act as a transport medium, such as in the blood of animals and in the sap in the xylem
of plants.
2. High specific heat capacity: Water has a high specific heat capacity. This means that a large amount of heat is
required to increase the temperature of 1kg of water by 1°C (1K). This is because most of the heat is used in
breaking hydrogen bonds, which restrict the movement of the molecules.
The result of this property is that water acts as a temperature buffer, that is, it minimises temperature changes
wherever it is present. Biochemical processes can, therefore, take place within a small temperature range, and
are less likely to be inhibited by extremes of temperature. Water also provides a very constant external
environment for many cells and organisms.
3. High latent heat of vaporization: Water has a high latent heat of vaporization, meaning that a large amount of
heat is required to water from its liquid state at 100°C to vapour at the same temperature. This is also as a
result of hydrogen bonding, and results in the unusually high boiling point of water. The energy transferred to
water to vaporize it results in the loss of energy (in the form of heat) from its surroundings. This results in the
cooling of the body from which the energy (heat) was removed.
This property is made use of in the process of sweating and panting in mammals, and may also be important in
the cooling of transpiring leaves. The high latent heat of vaporization also means that a large amount of heat
can be lost with a minimal loss of water from the body.
4. Density and freezing properties: Water is most dense at 4°C. Below this temperature, the density decreases
and, therefore, ice floats on water. Thus, water is the only substance whose solid form is less dense than its
liquid form.
Since ice floats on water, it means that bodies of water freeze from the top downwards. If ponds, for example,
froze from the bottom upwards, freshwater life would not exist in temperate or arctic climates. Ice at the
surface insulates the water below it, increasing the chances of survival below the surface. Also when the
temperature increases, the ice also thaws fast because it is at the surface.

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5. High latent heat of fusion: Water has a high latent heat of fusion, that is, a large amount of heat is required to
convert ice at 0°C to water at the same temperature. Conversely, it means that water must lose a relatively
large amount of heat in order for it to freeze.
This means, therefore, that cell contents and their environments are less likely to freeze in cold weather. Ice
crystals are particularly damaging if they form within calls.
6. High surface tension and cohesion: cohesion is the force by which individual molecules of a substance stick
together. At the surface of a liquid, there is a force called surface tension, which exists as a result of cohesive
forces between the molecules. This causes the surface of the liquid to occupy the smallest possible space
(which, ideally, would be a sphere). Water has a higher surface tension than many other liquids.
The high cohesion of water is important in cells, and in the translocation of water through the xylem in plants.
Also, many organisms depend on the surface tension to settle on water or to move over its surface.

1.3 THE SIGNIFICANCE OF CARBON IN BIOLOGICAL MOLECULES

1.4 THE CARBOHYDRATES

Carbohydrates are polyhydroxyl derivatives of alcohols; that is, they are alcohols with many hydroxyl groups. They
have the general formula CxH2yOy. They include the monosaccharides, disaccharides and polysaccharides. Sugars
and some polysaccharides (starch and glycogen) serve as energy storage compounds. Another polysaccharide is
cellulose, which is the most abundant organic molecule on earth because it supports plant cell wall. Carbohydrates
are also attached to the surfaces of animal cells, and they are specific to the individual.

Monosaccharides are simple sugars with a carbon backbone of 3-9 carbon atoms. The best known are the hexoses,
such as glucose in the blood of animals, and fructose found in fruits. They both have the formula C 6H12O6, but differ
in structure [find out their structures]. Ribose and deoxyribose are pentoses (C 5H10O5) of significant function
because they are found in the nucleic acids.

Disaccharides are made up of two monosaccharides joined together by a condensation reaction. Lactose is made
up of glucose and galactose, and it is found in milk. Maltose is made up of two glucose molecules, and it is a by-
product of starch digestion. Sucrose is made up of glucose and fructose, and it is the table sugar that we use, which
is produced by plants such as sugar cane and sugar beets [Look up the reactions and structures].

Polysaccharides contain many sugar units joined by condensation. Starch and glycogen are made up of many α-
glucose molecules. They are found in plants and animals, respectively. They serve the purpose of storage, and can
be readily broken down to provide energy for metabolism. Cellulose and chitin are made up of β-glucose molecules,

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and are found in plants and animals, respectively. They serve a structural purpose and cannot be broken down
easily.

Assignment: Write an essay on isomerism in carbohydrates, highlighting the significance of the various
categories of isomers.
1.5 LIPIDS
This group is made up of a variety of compounds which have no general formula. They share the general property
of being insoluble in water because they lack polar groups. Like carbohydrates, they contain carbon, hydrogen and
oxygen, but the proportion of oxygen is much lower than in carbohydrates. Some lipids also contain phosphorus.
The most familiar lipids are the fats and oils. Fats are solid at room temperature and are generally found in animals,
whereas oils are liquid at room temperature and are generally found in plants. Both groups of compounds are
formed from fatty acids and glycerol [How?]. They differ in the types of fatty acids that they contain, and whether
the fatty acids are saturated or not. Fats contain mainly saturated fatty acids, while oils contain mainly unsaturated
fatty acids. The greater the proportion of saturated fatty acids, the more likely the lipid is to be solid, whereas it
tends to be liquid when the proportion of fatty acids is low. Fats are used by most animals for long-term storage of
energy. Weight by weight, they produce more energy on oxidation than carbohydrates. Along with oils, fats serve
as good insulators, especially for animals in cold climates.

Phospholipids and steroids are also important lipids found in living things. For example, they are components of the
plasma membrane, which encloses cells. Phospholipids are lipids containing a phosphoric acid residue. The portion
of the molecule with the phosphate group is polar (hydrophilic), while the rest of the molecule is non-polar
(hydrophobic). Such a molecule is said to be amphipathic. Phospholipids form a double layer in cell membranes.
Steroids have a backbone of four fused carbon rings, but differ from each other by the type of functional group
attached to the rings. Cholesterol is a steroid, and it is also a precursor of other steroids, such as the hormones
oestrogen and testosterone.

1.6 PROTEINS
A protein is composed of one or more polypeptides, which are polymers of amino acids. Amino acids have the
general formula: H and differ according to their R groups.
H 2N-C-COOH
R
There are about 20 naturally occurring amino acids. [Look up the names and structures of these amino acids.]

Amino acids form peptides by condensation reactions between the carboxylic group (-COOH) of one amino acid and
the amino group (H2N-) of the other. Because of their spatial arrangements, the one amino acid has to be inverted

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(turned upside down) relative to the other. The peptide formed has a free amino group at one end, and a free
carboxylic group at the other (while one R group points upwards and the other points downwards). Thus, the
peptide can be lengthened by adding more amino acids to these free groups.
Proteins serve several functions which include:
 supporting structures, such as nails, hair and collagen
 enzymes, which catalyse biochemical reactions at room temperature
 transport proteins, such as channel proteins and carrier proteins on cell membranes
 defence, as in antibodies present in human blood
 hormones, which are regulatory proteins
 motion, by means of actin and myosin fibres found in muscles.

There are four levels of structural organization in proteins. However, not all proteins have all four levels. The
primary (1°) structure deals with the sequence of amino acids joined by peptide bonds. The secondary (2°) structure
occurs when segments of a polypeptide coil or fold in a particular way. The tertiary (3°) structure is the folding and
twisting that results in the overall three-dimensional shape of the polypeptide. The quaternary (4°) structure occurs
in proteins consisting of more than one polypeptide chain, and it deals with how the individual polypeptides are
folded around each other.

The sequence of amino acids in a polypeptide determines it final shape, because various R groups interact
differently. The function of a protein is dependent upon its shape.

1.7 NUCLEIC ACIDS

Nucleic acids are polymers of nucleotides, and they perform specific functions in cells. A nucleotide is made up of a
nitrogenous base, a pentose sugar, and at least one phosphate group. Some nucleotides have independent
metabolic functions in cells. For example, some are components of coenzymes, which facilitate enzymatic
reactions. ATP is a nucleotide that supplies energy for synthetic reactions, and any other energy-requiring process
in cells.

DNA is the genetic material that stores information regarding its own replication, and the order in which amino
acids are arranged to form a protein. RNA is another type of nucleic acid, and it conveys information from DNA to
the sites of protein synthesis. In DNA, the pentose sugar is deoxyribose, whereas in RNA it is ribose. There are four
types of nitrogenous bases in DNA and four in RNA. The bases in DNA are adenine, guanine, cytosine and thymine,
whereas in RNA they are adenine, guanine, cytosine and uracil. Thus, adenine, guanine and cytosine are common to
both, while thymine is unique to DNA and uracil is unique to RNA. Adenine and guanine are purines, which are

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nitrogenous bases having a double ring, while cytosine, thymine and uracil are pyrimidines, which are nitrogenous
bases having a single ring.

DNA is double-stranded, with the two strands usually twisted around each other to form a double helix. The two
strands are held together by hydrogen bonds between the purines and pyrimidines. The bases can occur in any
order within a strand, but between strands adenine always binds with thymine and guanine binds with cytosine.
This is called complementary base pairing. Therefore, in any DNA molecule, the number of adenine always equals
the number of thymine, and the number of guanine always equals that of cytosine.
RNA is single-stranded.

1.8 ATP (ADENOSINE TRIPHOSPHATE) - THE ENERGY CURRENCY OF THE CELL

ATP is a nucleotide composed of adenine, ribose and three phosphate groups. It is a high energy molecule because
the two phosphate bonds are unstable high-energy bonds, which are easily broken. Usually, in cells the terminal
bond is hydrolysed to give ADP and inorganic phosphate.

The energy released from ATP breakdown is coupled to energy-requiring processes like the synthesis of
macromolecules such as carbohydrates and proteins. In muscle cells, the energy is used for muscle contraction, and
in nerve cells, it is used for the conduction (transmission) of nervous impulses. ATP is used up (hydrolysed) to
provide energy when needed by cells, and when energy is released in cells, it is converted to ATP. Therefore, ATP is
known as the energy currency of cells.

The particular importance of ATP in biological systems lies in its three phosphate groups. One of these phosphate
groups is connected to the ribose via an ester linkage, but the other two are coupled to each other and to the third.
Phosphates linked in this way are called pyrophosphates and form as a result of the elimination of water between
two phosphoric acid units.

ATP can act as an intermediary by virtue of the fact that the removal of a phosphate group by hydrolysis is a highly
exothermic reaction. The products of hydrolysis are ADP, an inorganic phosphate and energy.

ATP + H2O ⇌ ADP + Pi + energy

The reaction above has a tendency to proceed from left to right, because the equilibrium constant is high, but the
reaction takes place at an appreciable rate only in the presence of appropriate catalysts. The reaction from right to
left occurs during photosynthesis and catabolism of food materials. The reaction is not spontaneous, but can
proceed if coupled with highly exothermic reactions, so that the net result is also exothermic. The essence is in

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organizing energy-producing and energy-requiring reaction sequences such that ATP formation or ATP hydrolysis
occur during the course of the sequences.

Strictly speaking, ATP is not stored, and is not transported between cells. If there is excess energy-yielding capacity,
such as just after a meal, the ATP is then invested by using it to drive the synthesis of storage materials such as fats
and glycogen.

ENZYMES
2.1 INTRODUCTION
An enzyme is a biological molecule (protein) that serves as a catalyst for a biochemical (metabolic) reaction.
Enzymes are proteins. However, several RNA molecules have been shown to have the ability to catalyse biological
reactions. They are known as ribozymes.

Without enzymes to speed up biochemical reactions, life could not exist, because the life of cell depends upon the
simultaneous occurrence of thousands of chemical reactions that must take place rapidly under mild conditions. An
enzyme facilitates a biochemical reaction by lowering the activation energy and increasing the rate of the reaction.

The bases of enzyme activity are high specificity and rapid reaction rates. A typical cell contains several thousand
different molecules, and each is important to the chemistry of life processes. Each enzyme recognizes only one, or
sometimes a few, of these molecules. This specificity enables an enzyme to recognize and bind with a substrate and
transform it to product at an incredible speed. In fact, enzyme-catalysed reactions often occur at rates between
one million and one hundred million times faster than the uncatalysed reaction. In effect, without enzymes,
metabolic reactions would occur at a rate that is too slow to sustain life.

2.2 PROPERTIES OF ENZYMES

1. Enzymes are protein in nature, and they are coded for by DNA.
2. Being proteins, enzymes are affected by the factors that affect proteins, such as extremes of heat and
temperature.
3. Every enzyme works at its fastest rate within a narrow optimum range of conditions such as pH and
temperature.
4. An enzyme is several times larger than the substrate upon which it acts.
5. An enzyme has several thousand active sites for its substrate(s). The active sites have specific shapes that are
complementary to the structure of the substrate(s).
6. Enzymes lower the activation energy of reactions by bringing reactants into close proximity with each other,
such that less energy is required is required for successful collisions between the reactants to form products.
7. The reactions enzymes catalyse are usually reversible.

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8. Enzymes remain unchanged at the end of reactions they catalyse, and their presence does not alter the nature
or properties of the products of their reactions.

2.3 CLASSIFICATION OF ENZYMES

Enzymes can be classified according to the type of reaction they catalyse. Six classes of enzymes are recognized as
follows:
i. Oxidoreductases - these are enzymes that catalyse redox reactions. They can be subdivided into oxidases and
reductases. An example is lactate dehydrogenase, which is an oxidoreductase that removes hydrogen from a
molecule of lactate.
ii. Transferases - these enzymes catalyse the transfer of functional groups from one molecule to another. For
example, a transaminase catalyses the transfer of an amino functional group, and a kinase catalyses the transfer of
a phosphate group.
iii. Hydrolases - these enzymes catalyse hydrolysis reactions, that is, the addition of a water molecule to a bond,
resulting in bond breakage. These reactions are important in the digestive process. For example, lipases catalyse
the hydrolysis of ester bonds in triglycerides.
iv. Ligases - these enzymes catalyse a reaction in which a -C-C-, -C-S-, -C-O- or -C-N- bond is made or broken. This is
often accompanied by an ATP-ADP inter-conversion. For example, DNA-ligase catalyses the joining of the OH group
of a DNA strand with the phosphoryl group of the adjacent group to form a phosphodiester bond.
v. Isomerases - these enzymes rearrange the functional groups within a molecule, and catalyse the conversion of
one isomer to another. An example is phosphoglycerate mutase which converts 3-phosphoglycerate into 2-
phosphoglycerate.
vi. Lyases - these catalyse the addition of a group to a double bond, or the removal of a group from a double bond.
An example is carbonic anhydrase, which catalyses the reversible reaction between carbon dioxide and water in the
blood to form carbonic acid. This reaction is one of the body’s mechanisms for buffering body fluids. Another
example is citrate lyase, which catalyses the removal of an acetyl group from a molecule of citrate.

2.4 THE EFFECTS OF ENZYMES ON REACTIONS

An enzyme changes the pathway by which a reaction occurs. It provides a lower energy route for the conversion of
the substrate into the product. An enzyme, therefore, speeds up a reaction by lowering the activation energy of the
reaction.

If we consider an equilibrium reaction: aA ⇌ bB, the equilibrium constant Keq is given as:
Keq = [B]b

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 [A]a
This Keq is a reflection of the difference in energy between the reactant(s) and product(s). No matter how the
reaction occurs, the energy difference between the reactants and products is always the same. An enzyme,
therefore, does not alter the equilibrium constant for the reaction it catalyses. However, the enzyme does change
the pathway by which the process occurs, and consequently increases the rate at which the reaction reaches
equilibrium.

For uncatalysed chemical reactions, the rate often doubles as the substrate concentration is doubled. Therefore, as
long as the substrate concentration increases, there is a direct increase in the rate of the reaction. For enzyme-
catalysed reactions, however, this is not the case. The rate of the reaction initially increases with an increase in the
substrate concentration, but at a certain concentration the reaction reaches its maximum rate. At this maximum
rate, all the active sites of all the enzyme molecules in solution are occupied by substrate molecules. No new
substrate molecule can bind on to any active site of any enzyme molecule except the substrate occupying that
active site has been converted to product and released. The reaction rate is, therefore, dependent upon the
availability (concentration) of the enzyme. If the enzyme concentration increases, there would again be an initial
increase in the reaction rate until the active sites are saturated, after which there would be no further increase in
the reaction rate.

2.5 ENZYME-SUBSTRATE INTERACTION

The chemical upon which an enzyme works is its substrate. An enzyme combines with its substrate to form a short-
lived enzyme-substrate complex. The proximity of the enzyme with the substrate greatly increases the chances of a
reaction occurring. Once a reaction has occurred, the complex breaks up into product(s) and enzyme. The enzyme
remains unchanged at the end of the reaction, and is free to interact with more substrates.
E + S ⇌ ES ⇌ EP ⇌ E + P
The rate of an enzymatic reaction is the rate at which the substrate disappears, and at which the product is being
formed.

Enzymes speed up reactions by lowering the activation energy of those reactions. They do this by bringing the
reactants close together, and by stretching their intra-molecular bonds. The substrates fit into the active sites,
which are small portions of the enzyme. The stretching of the bonds makes it easy for them to be broken, and for
new bonds to be formed.

The active site, into which the substrate fits, has the following characteristics:
- it is a cleft or pocket in the surface of the enzyme;
- the R-groups in the active site that are involved in catalysis are called catalytic groups;

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- the shape of the active site is complementary to the shape of the substrate, that is, the substrate fits neatly into
the active site;
- an enzyme attracts and holds on to its substrate by weak, non-covalent interactions. The R-groups involved in
substrate binding, but not necessarily in catalysis, make up the binding site (groups).
- the conformation of the active site determines the specificity of the enzyme, because only the molecule
(substrate) that fits into the active site would be used in a reaction.

A hypothesis proposed by Fischer (1890) suggested that the active site of the enzyme has a specific shape into
which the substrate exactly fits. This is referred to as the lock and key hypothesis, wherein the substrate is like a
key which fits into the complementary shape of the active site or lock. Each active site is made up of 3-12 amino
acids, while the remainder of the enzyme serves to maintain its proper three-dimensional shape for the effective
functioning of the active sites. Once products are formed, they can no longer fit into the active site, and are
released into the surrounding medium, leaving he enzyme free to receive more substrate molecules.

A modification of the lock and key hypothesis, known as the induced-fit hypothesis, was proposed by Koshland
(1958). He suggested that some enzymes and their active sites were more flexible, and that the active site could be
modified as the substrate interacts with the enzyme. The amino acids which make up the active site could be
moulded into a precise shape which enables the enzyme to perform its catalytic function effectively. In such cases,
it is believed that the substrate molecule changes shape slightly as it enters the active site. Thus, the shape of the
enzyme and substrate are changed slightly in order to obtain conformity and ensure a ‘perfect fit’.

2.6 ENZYME SPECIFICITY

The requirement for a perfect fit determines whether an enzyme will bind to a particular substrate and carry out a
chemical reaction. Enzyme specificity is the ability of an enzyme to bind to only one substrate, or very few
substrates, and thus catalyse only a single reaction, or a single type of reaction. For example the enzyme urease
catalyses the breakdown of urea to carbon dioxide and ammonia, but does not act upon methyl urea, even though
it is structurally similar to urea.

Not all enzymes demonstrate the same degree of specificity. Four classes of enzyme specificity have been
observed:
 Absolute specificity - this is shown when an enzyme catalyses the reaction of only one substrate. Aminoacyl t-
RNA synthetases exhibit absolute specificity. Each of them must attach the correct amino acid to the correct t-
RNA molecule. If the wrong amino acid is attached to the t-RNA, it would mistakenly be attached to the peptide
chain, producing a non-functional protein.

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 Group specificity - in this case, an enzyme catalyses processes involving similar molecules having the same
functional group. Hexokinase is a group-specific enzyme that catalyses the addition of a phosphoryl group to
glucose (a hexose) in the first step of glycolysis. It can also add a phosphoryl group to other hexoses.
 Linkage specificity - this occurs when an enzyme catalyses the formation or breakage of only certain bonds in a
molecule. Examples are the proteases such as trypsin, chymotrypsin and elastase, which selectively hydrolyse
peptide bonds.
 Stereochemical specificity - this is the situation wherein an enzyme can distinguish one enantiomer
(stereoisomer) from another. Most of the enzymes in the human body show this specificity. Because we use only
D-sugars and L-amino acids, the enzymes involved in digestion and metabolism recognize only those particular
stereoisomers.
2.7 COFACTORS AND COENZYMES
Some enzymes need an additional non-protein prosthetic group in order to function. The polypeptide portion of
such an enzyme is called the apoenzyme, while the non-protein prosthetic group is called the cofactor. Together,
they make up the active enzyme called the holoenzyme. Cofactors may be metal ions, organic compounds or
organometallic compounds. They must be bound to the enzyme to maintain the correct configuration of the active
site. Without the cofactor, the enzyme cannot bind to the substrate and catalyse its reaction.

Other enzymes require binding temporarily with a coenzyme in order for them to function properly. A coenzyme is
a low molecular weight organic molecule that generally serves as a carrier of electrons or chemical groups. In
biochemical reactions, they may either donate groups to the substrate, or serve as recipients of groups that are
removed from the substrate. The coenzyme is often bound to the enzyme by weak interactions like hydrogen
bonds. Often, the coenzymes contain modified vitamins as part of their structure. Of the water-soluble vitamins,
only vitamin C has not been associated with a coenzyme. Examples of coenzymes include NAD and NADP, which are
derived from niacin, and FAD, which is made from riboflavin.

2.8 INHIBITORS
Inhibitors are chemicals that can bind to enzymes and either eliminate or drastically reduce their catalytic ability.
Examples include salts of heavy metals such as arsenic and mercury, and drugs such as penicillin. Inhibitors are
classified according to whether their inhibition is reversible or irreversible, and competitive or non-competitive.
Reversibility deals with whether the inhibitor will eventually dissociate from the enzyme, releasing it in the active
form. Competition refers to whether the inhibitor is a structural analogue of the substrate.
 Irreversible inhibitors - such inhibitors (for example arsenic) usually bind very tightly, sometimes even covalently,
to the enzyme. This generally involves the binding of the inhibitor to one of the R-groups of the amino acids in
the active site. The inhibitor may block the binding groups of the active site, so that the enzyme-substrate
complex cannot form. Alternatively, the inhibitor may interfere with the catalytic groups in the active site,

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 thereby effectively eliminating catalysis. Irreversible inhibitors generally inhibit many different enzymes. Other
examples include snake venoms and nerve gases.
 Reversible competitive inhibitors - these are often structural analogues of the natural substrate of the enzyme,
that is, they resemble the structure and charge distribution of the substrate. Because of this similarity, the
inhibitor can occupy the same active site as the substrate. However, no reaction can occur, but the enzyme’s
activity will be inhibited. This inhibition is competitive because the inhibitor and substrate compete for the same
active site. The degree of inhibition, therefore, depends on their relative concentrations. If the inhibitor is in
excess or binds more strongly to the active site, it would occupy the active site more frequently. On the other
hand, if the natural substrate in present in excess, it will more frequently occupy the active site, and there would
be little inhibition. Examples include the sulfa-drugs.
 Reversible non-competitive inhibitors - these inhibitors bind to the R-groups of amino acids, or sometimes to the
metal ion cofactors. Unlike irreversible inhibition, the binding is weak and the enzymatic activity is restored when
the inhibitor dissociates from the enzyme-inhibitor complex. Although the inhibitor does not generally bind to
the active site, it does modify the shape of the active site by binding elsewhere on the enzyme. Because the
activity of the enzyme is dependent upon maintaining the correct shape, if the shape is altered by binding
elsewhere with the inhibitor, catalysis of the reaction involving the substrate would be inhibited.

2.9 REGULATION OF ENZYME ACTIVITY

One of the major ways in which enzymes differ from non-biological catalysts is that the activity of the enzyme is
often regulated by the cell. There are various reasons for this control of enzyme activity. Some reasons involve
energy conservation. The cell has many mechanisms to conserve energy, because if the cell runs out of energy, it
will die. For example, it would be a great waste of energy to produce an enzyme if its substrate is not available.
Similarly, if the product of an enzymatic reaction is in excess, it would be a waste of energy to continue producing
more of it.

The simplest mechanism to regulate enzyme activity is to produce the enzyme only when the substrate is present.
Other mechanisms include the use of allosteric enzymes, feedback mechanism, production of zymogens and
protein modification.

METABOLISM
3.1 INTRODUCTION
Metabolism is the sum of all reactions that go on in a living organism. There are thousands of reactions going on in
a given cell at any point in time, and these reactions are catalysed by specific enzymes. The substrates which
participate in metabolism are known as metabolites, and they are also the substrates and products of the enzymes.

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Metabolism can be divided into catabolism and anabolism. Catabolism is the process by which complex materials
are broken down. Catabolic reactions serve a dual purpose: (i) they produce the raw materials or building blocks for
building new cellular materials, and (ii) they also provide the energy for these building processes. Anabolic
reactions are reactions in which complex molecules are built up from simpler molecules. Catabolic reactions tend to
be exothermic (exergonic), while anabolic reactions tend to be endothermic (endergonic).

Organisms have a need to take in energy, and to utilise it. Autotrophic organisms utilise sunlight or chemical energy
to build up their required macromolecules, including their food stores. They also break down these food stores to
release energy for other cellular processes. Heterotrophs obtain their energy from foods already manufactured by
autotrophs. They break down these foods to produce energy, and for raw materials to make up their own
macromolecules.

Metabolic reactions do not proceed in a single step, but involve a series of steps. This is because
 the total energy required by anabolic reactions, or given off by catabolic reactions, would be too great for
the cell to tolerate, and would lead to the death of the cell if taken in or released in a single-step reaction;
 several enzymes are involved in anabolic or catabolic reactions, and each has its own optimum conditions;
 the products of one step of a reaction become the inputs of another reaction.

For these purposes, anabolic and catabolic reactions are coupled in biological systems, so that the products of one
step of a reaction are used in another reaction. Furthermore, energy is released from energy-rich macromolecules
in small bits for efficient use, and to prevent wastage. [Read about Cellular Respiration and Photosynthesis. These
entail Basic Metabolism.]

3.2 THE PENTOSE PHOSPHATE PATHWAY

The pentose phosphate pathway (PPP) is an alternative pathway for the oxidation of glucose. It is also known as the
Warburg-Dickens pathway, the phosphogluconate pathway, or the hexose monophosphate shunt. While the
pentose phosphate pathway does the involve oxidation of glucose, its primary role is anabolic rather than catabolic.
This pathway is important for the following reasons:
 organisms have a need for biosynthetic capacities beyond those represented by intermediates of the
glycolytic sequence;
 there is also a need for a source of reducing power in the biosynthetic processes. This reducing power is
produced in the form NADPH.

13
The pathway generates NADPH and pentoses (5-carbon sugars), as well as ribose 5-phosphate, a precursor for the
synthesis of nucleotides. The pentose phosphate pathway is most active in tissues involved in cholesterol and fatty
acid syntheses. These two processes require abundant NADPH. Consequently, very high levels of PPP enzymes are
present in the liver, which is the site of cholesterol synthesis and a major site of fatty acid synthesis, and in adipose
tissue, where fatty acid synthesis also occurs. The pathway is also especially important in red blood cells
(erythrocytes). For most organisms, the pentose phosphate pathway takes place in the cytosol, but in plants, most
steps take place in plastids.

The PPP is a very complex pathway, but it can be considered to consist of three main stages. The first stage is the
oxidative stage, which is shown below:

Glucose-6-phosphate (1) 6-phosphoglucono-δ-lactone (2) 6-phosphogluconate (3) ribulose-5-

phosphate (4). In this phase, two molecules of NADP + are reduced to NADPH, utilizing the energy from the
conversion of glucose-6-phosphate into ribulose 5-phosphate. These reactions produce the NADPH required for
biosyntheses. The reaction is summarized below:
Glucose-6-phosphate + 2NADP + + H2O ribulose 5-phosphate + 2NADPH + 2H+ + CO2

The second stage involves isomerism reactions that convert ribulose-5-phosphate to ribose-5-phosphate or
xylulose-5-phosphate. The pathway’s name reflects the production of these phosphorylated five-carbon sugars
(that is, pentose phosphates).

The third stage is a complex series of reactions involving C-C bond cleavage and formation. The result of these
reactions is the formation of two molecules of fructose-6-phosphate and one molecule of glyceraldehyde-3-
phosphate from three molecules of pentose phosphates. The two stages are summarized below:
3 ribulose-5-phosphate ribose-5-phosphate + 2 xylulose-5-phosphate 2 fructose-6-phosphate +
glyceraldehyde-3-phosphate
In addition to providing reducing power, the PPP provides sugar phosphates that are required for biosynthesis. For
example, ribose-5-phosphate is used in the synthesis of nucleotides such as ATP. Also, the four-carbon sugar
phosphate, erythrose-4-phosphate, produced in the third stage of the pentose phosphate pathway is a precursor of
the amino acids phenyl-alanine, tyrosine and tryptophan.

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3.3 FATTY ACID DEGRADATION
The metabolism of fatty acids and lipids revolves around the fate of acetyl coenzyme A. Under aerobic conditions,
pyruvate formed from glucose is converted to acetyl coenzyme A (acetyl co-A). Similarly, fatty acids are also
degraded to acetyl coenzyme A and oxidized by the citric acid cycle. Moreover, acetyl coenzyme A is itself the
starting material for is the biosynthesis of cholesterol and steroidal hormones. Acetyl coenzyme A is thus a key
intermediary in lipid metabolism. Beta (β)-oxidation releases acetyl-CoA, FADH2 and NADH, the three of which
enter the citric acid cycle or Krebs’ cycle, in which ATP is produced to be used as energy. The process continues

15
until two acetyl-CoA molecules are produced and the fatty acyl-CoA chain has been completely broken down. In
eukaryotic cells, β-oxidation takes place in the mitochondria, whereas in prokaryotic cells, it happens in the cytosol.
In cases where fatty acid chains are too long to enter the mitochondria, the process can also take place in
peroxisomes.

A German scientist, Franz Knoop, working with labelled fatty acids, called omega (ω) labelled fatty acids, found out
that when the fatty acid had an even number of carbon atoms in the chain, phenyl acetate was formed, but when it
had an odd number, benzoate was formed. He interpreted this to mean that the degradation of fatty acids occurs
by the removal of two-carbon acetate groups from the COO - end of the fatty acid. These two-carbon fragments are
not actually acetate but acetyl coenzyme A. The pathway for the breakdown of fatty acids into acetyl coenzyme A is
called β-oxidation.

The β-oxidation cycle (steps 2 to 5) consist of a set of four reactions whose overall form is similar to the last four
reactions of the citric acid cycle. Each trip through the sequence of reactions releases acetyl coenzyme A and
returns a fatty acyl coenzyme A molecule that has 2 less carbon atoms. One molecule each of FADH 2 and NADH are
produced for each cycle of β-oxidation.

3.3.1 The reactions of β-oxidation

The enzymes that catalyze the β-oxidation of fatty acids are located in the matrix of the mitochondrion. Special
transport mechanisms are required to bring fatty acids into the mitochondrial matrix. Once in the matrix, the fatty
acids are degraded, and the reactions involved (that is, β-oxidation) interact with oxidative phosphorylation and the
citric acid cycle to produce ATP.

Reaction 1: The first step is an activation reaction that results in the production of a fatty acyl coenzyme A
molecule. A high-energy thioester bond is formed is formed between the fatty acid and coenzyme A. The energy for
this reaction is provided by ATP.

Reaction 2: The next step is an oxidation reaction that removes a pair of H-atoms from the fatty acid. These are
used to reduce FAD to FADH2.

Reaction 3: This reaction involves the hydration of the double bond produced in reaction 2. As a result, the β-
carbon is hydroxylated.

Reaction 4: This is an oxidation reaction in which the OH-group of the β-carbon is dehydrogenated. NAD + is reduced
to NADH.

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Reaction 5: This final reaction is the cleavage that releases acetyl coenzyme A. This is accomplished by thiolysis, that
is, the attack of a molecule of coenzyme A on the β-carbon. The result is the release of acetyl coenzyme A and a
fatty acyl coenzyme A that is two carbon atoms shorter than the starting fatty acid.
The shortened fatty acyl coenzyme A is further oxidized by cycling through reactions 2-5 until the fatty acid carbon
chain is completely degraded to acetyl coenzyme A. The acetyl co A produced by β-oxidation of fatty acids then
enters the citric acid cycle. This eventually results in the production of 12 ATP molecules per mole of coenzyme A.

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3.4 DEGRADATION OF AMINO ACIDS - Reading assignment.
 Amino acid breakdown can yield:
o Acetyl-CoA
o α −KG
o Succinl-CoA
o 0AA
o Fumarate
a-KG is generated from five amino acids

 Proline
 Glutamate
 Glutamine
 Arginine
 Histidine

Not a surprise

 Proline, glutamate, arginine, and glutamate are synthesized from a-KG, here use distinct enzymes
for breakdown
 But, histidine is not A completely different pathway for histidine catabolism than for anabolism,
 In this case, incoming amino acid (glutamate) binds, donates its amino group to phosphate, and
leaves as an a-keto acid (a-KG). Then, an incoming a-deto acid binds and accepts the amino group
and leaves as an amino acid

Four amino acids are converted to Succinyl-CoA

 Methionine
 Converted to homocysteine through methyl group transfer, generates cysteine as converted to
a-ketobutyrate
 Isoleucine
 Transamination, oxidative decarboxylation to acetyl-CoA and propionyl CoA
 Valine
 Transamination, decarboxylation to propionyl CoA
 Threonine
 a-ketobutyrate generated and converted to propionyl CoA

Propionyl-CoA is a common intermediate for amino acids? succinyl-CoA

Branched-chain a-keto acid dehydrogenase complex

 In certain body tissues, this enzyme catalyzes the oxidative decarboxylation of valine,
isoleucine, and leucine yielding CO2, and acyl-CoA derivatives.
 Shares ancestry with pyruvate dehydrogenase complex, a-KG dehydrogenase complex
another example of gene duplication

Branched-chain complex Asparagine and aspartate are degraded to OAA

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Fate of metabolites derived from amino acids

 In addition to feeding the citric acid cycle, amino acids can result in ketone bodies, while
others are gluconeogenic

Ketone bodies

 The six amino acids that are degraded to acetoacetyl-CoA and/or acetyl-CoA (in blue on
previous slide) can be converted to acetoacetate and b-hydroxybutyrate

Gluconeogenic amino acids

 Amino acids that are degraded to pyruvate, a-KG, succinyl-CoA fumarate, and/or OAA can
be converted to glucose

Tempting to take a dietary perspective on carbohydrate and protein metabolism, but Well just re-
emphasize ammonia metabolism You seen this many, many times

 All aminotransferases have PLP

PLP enzymes

 Generally found in enzyme active site covalently

bound to amino group of lysine PLP-mediated transformation
Aminotransferases exhibit Ping-Pong kinetics

 Ping-pong no ternary complex is formed between substrates and enzyme first substrate
binds, reacts, then that products leaves before second substrate binds

Ammonia from amino acid catabolism

 During amino acid breakdown, amino is generally transferred to

 glutamate (serves as nitrogen
 source and sink)
 From there, amino group can be
 released as ammonia

Transdeamination

 The combined action of aminotransferase and glutamate dehydrogenase is called

transdeamination
 Glutamate dehydrogenase operates at an important intersection of carbon and nitrogen
metabolism as a result, highly regulated

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Linkage of TCA with aa catabolism by allostery

 ADP is a positive effector of glutamate dehydrogenase, while GTP is a inhibitor

Ammonia is toxic, so cells need to get rid of it

 Fix ammonia onto glutamate to form glutamine and use as a transport mechanism
 Transport ammonia by glucose-alanine cycle
 Excrete nitrogenous waste through urea cycle
 Glucose-alanine cycle

Ammonia transport using alanine

 Alanine aminotransferase transfers the a-amino group from glutamate to pyruvate,

forming alanine
 This shuttle funnels ammonia out of tissues that have high glycolytic flux, to the liver,
which can remove ammonia via urea cycle

Dumping ammonia as urea

 The glutamine, glutamate, and alanine feed the urea cycle

 The urea cycle generates urea, which can be deposited as waste
 The urea cycle spans both the cytosol and mitochondria and four intermediates you are
responsible for the urea cycle

3.5 THE UREA CYCLE - READING ASSIGNMENT

The urea cycle is the first metabolic pathway to be elucidated.
 The cycle is known as Krebs–Henseleit urea cycle.
 Ornithine is the first member of the reaction; it is also called as Ornithine cycle.
 Urea is synthesized in liver & transported to kidneys for excretion in urine.
 The two nitrogen atoms of urea are derived from two different sources, one from ammonia & the other
directly from the amino group of aspartic acid.
 Carbon atom is supplied by CO2
 Urea is the end product of protein metabolism (amino acid metabolism).
 Urea accounts for 80-90% of the nitrogen containing substances excreted in urine.
 Urea synthesis is a five-step cyclic process, with five distinct enzymes.
 The first two enzymes are present in mitochondria while the rest are localized in cytosol.

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STEP 1: Formation of carbamoyl phosphate.
 Carbamoyl phosphate synthase I (CPS I) of mitochondria catalyses the condensation of
NH4 + ions with CO2 to form carbamoyl phosphate.
 This step consumes two ATP & is irreversible.
 It is a rate-limiting.
 CPS I requires N-acetylglutamate for its activity.
 Carbamoyl phosphate synthase II (CPS II) -
involved in pyrimidine synthesis & it is present
in cytosol.
 It accepts amino group from glutamine & does not require N-acetylglutamate for its activity.
STEP 1: formation of carbamoyl phosphate

Carbamoyl Phosphate synthesis

CPS-I CPS-II
 Mitochondria  Cytosol
 Uses NH3  Uses Glutamine
 Pyrimidine biosynthesis
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 Inhibited - CTP
 Urea Cycle
 Activated – NAG

STEP 2: Formation of Citrulline

 The second reaction is also mitochondrial.
 Citrulline is synthesized from carbamoyl phosphate & ornithine by ornithine transcarbamoylase.
 Ornithine is regenerated & used in urea cycle.
 Ornithine & citrulline are basic amino acids. (Never found in protein structure due to lack of codons).
 Citrulline is transported to cytosol by a transporter system.
 Citrulline is neither present in tissue proteins nor in blood; but it is present in milk.

STRP 2: Formation of Citrulline

STEP 3: Formation of Arginosuccinate

 Citrulline condenses with aspartate to form arginosuccinate by the enzyme Arginosuccinate synthetase.
 Second amino group of urea is incorporated.
 It requires ATP, it is cleaved to AMP & PPi
 2 High energy bonds are required.
 Immediately broken down to inorganic phosphate (Pi).

STEP 4: Formation of Arginine or cleavage of Arginosuccinate

 The enzyme Argininosuccinase or argininosuccinate lyase cleaves arginosuccinate to arginine &
fumarate (an intermediate in TCA cycle)
 Fumarate provides connecting link with TCA cycle or gluconeogenesis.
 The fumarate is converted to oxaloacetate via fumarase & MDH & transaminated to aspartate.
 Aspartate is regenerated in this reaction.

NAD+ NADH+H+

Fumarate Malate Oxaloacetate Aspartate

Fumarase MDH Aminotransferase

STEP 5: Formation of Urea

 Arginase is the 5th and final enzyme that cleaves arginine to yield urea & ornithine.
 Ornithine is regenerated, enters mitochondria for its reuse in the urea cycle.
 Arginase is activated by Co2+ & Mn2+
 Ornithine & lysine compete with arginine (competitive inhibition).
 Arginase is mostly found in the liver, while the rest of the enzymes (four) of urea cycle are also present
in other tissues.
 Arginine synthesis may occur to varying degrees in many tissues.
 But only the liver can ultimately produce urea.

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Energetics of Urea Cycle
 The overall reaction may be summarized as:
 NH3 + CO2 + Aspartate → Urea + fumarate
 2ATPs are used in the 1st reaction.
 Another ATP is converted to AMP + PPi in the 3rd step, which is equivalent to 2 ATPs.
 The urea cycle consumes 4 high energy phosphate bonds.
 Fumarate formed in the 4th step may be converted to malate.
 Malate when oxidised to oxaloacetate produces 1 NADH equivalent to 2.5 ATP.
 So net energy expenditure is only 1.5 high energy phosphates.
 The urea cycle & TCA cycle are interlinked & it is called as "urea bicycle".

Disposal of urea
 Urea produced in the liver freely diffuses & is transported in blood to kidneys & excreted.
 A small amount of urea enters the intestine where it is broken down to CO2 & NH3 by the bacterial
enzyme urease.
 This ammonia is either lost in the feces or absorbed into the blood.
Disorders of the Urea cycle
 The main function of Urea cycle is to remove toxic ammonia from blood as urea.
 Defects in the metabolism of conversion of ammonia to urea, i.e., Urea cycle leads to
Hyperammonaemia or NH3 intoxication.

Hyperammoniaemia
 Inherited disorders of urea cycle enzymes - familial hyperammonaemia.
 Acquired disorders- Liver Disease, severe Renal disease - Acquired
hyperammonaemia.
Signs, symptoms and treatment
 CNS dysfunction or manifestations of failure of liver function (ascites, jaundice hepatomegaly, edema,
hemorrhage).
 The management of the condition is difficult.
 A low protein diet & intestinal disinfection (bowel clearing & antibiotics), withholding hepatotoxic
drugs & maintenance of electrolyte & acid-base balance.

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3.6 THE GLYOXYLATE CYCLE
The glyoxylate cycle, a variation of the tricarboxylic acid cycle, was discovered in 1957 at the University
of Oxford by Sir Hans Kornberg and his mentor Hans Krebs. It is an anabolic pathway occurring in plants,
bacteria, protists, and fungi, and it centers on the conversion of acetyl-CoA to succinate for the synthesis of
carbohydrates. In microorganisms, the glyoxylate cycle allows cells to utilize two-carbon compounds, such
as acetate, to satisfy cellular carbon requirements when simple sugars such as glucose or fructose are not
available. The cycle is generally assumed to be absent in animals, with the exception of nematodes at the
early stages of embryogenesis. In recent years, however, malate synthase (MS) and isocitrate lyase (ICL),
key enzymes involved in the glyoxylate cycle, have been discovered in some animal tissues. This has
raised questions regarding the evolutionary relationship of enzymes in bacteria and animals, and suggests
that animals encode alternative enzymes of the cycle that differ in function from known MS and ICL in
non-metazoan species.

Plants as well as some algae and bacteria can use acetate as the source for the production of carbon
compounds. Plants and bacteria employ a modification of the TCA cycle called the glyoxylate cycle to
produce four-carbon dicarboxylic acid from two-carbon acetate units. The glyoxylate cycle bypasses the
two oxidative decarboxylation reactions of the TCA cycle and directly converts isocitrate into malate and
succinate through ICL and MS.

Similarities with the tricarboxylic acid (TCA) cycle

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The glyoxylate cycle utilizes five of the eight enzymes associated with the TCA: citrate synthase,
aconitase, succinate dehydrogenase, fumarase, and malate dehydrogenase. The two cycles differ in that in
the glyoxylate cycle, isocitrate is converted into glyoxylate and succinate by isocitrate lyase (ICL) instead
of into α-ketoglutarate. This bypasses the decarboxylation steps that take place in the citric acid cycle
(TCA cycle), allowing simple carbon compounds to be used in the later synthesis of macromolecules,
including glucose. Glyoxylate is subsequently combined with acetyl-CoA to produce malate, catalyzed by
malate synthase. Malate is also formed in parallel from succinate by the action of succinate dehydrogenase
and fumarase.

Its role in gluconeogenesis

Fatty acids from lipids are commonly used as an energy source by vertebrates as fatty acids are degraded
through beta oxidation into acetate molecules. This acetate, bound to the active thiol group of coenzyme A,
enters the citric acid cycle (TCA cycle) where it is fully oxidized to carbon dioxide. This pathway thus
allows cells to obtain energy from fat. To utilize acetate from fat for biosynthesis of carbohydrates, the
glyoxylate cycle, whose initial reactions are identical to the TCA cycle, is used.

Cell-wall containing organisms, such as plants, fungi, and bacteria, require very large amounts of
carbohydrates during growth for the biosynthesis of complex structural polysaccharides, such as cellulose,
glucans, and chitin. In these organisms, in the absence of available carbohydrates (for example, in certain
microbial environments or during seed germination in plants), the glyoxylate cycle permits the synthesis of
glucose from lipids via acetate generated in fatty acid β-oxidation.

The glyoxylate cycle bypasses the steps in the citric acid cycle where carbon is lost in the form of CO 2. The
two initial steps of the glyoxylate cycle are identical to those in the citric acid cycle: acetate → citrate →
isocitrate. In the next step, catalyzed by the first glyoxylate cycle enzyme, isocitrate lyase, isocitrate
undergoes cleavage into succinate and glyoxylate (the latter gives the cycle its name). Glyoxylate
condenses with acetyl-CoA (a step catalyzed by malate synthase), yielding malate. Both malate and
oxaloacetate can be converted into phosphoenolpyruvate, which is the product of phosphoenolpyruvate
carboxykinase, the first enzyme in gluconeogenesis. The net result of the glyoxylate cycle is therefore the
production of glucose from fatty acids. Succinate generated in the first step can enter into the citric acid
cycle to eventually form oxaloacetate.

Function in organisms
- Plants

25
In plants the glyoxylate cycle occurs in special peroxisomes which are called glyoxysomes. This cycle
allows seeds to use lipids as a source of energy to form the shoot during germination. The seed cannot
produce biomass using photosynthesis because of lack of an organ to perform this function. The lipid stores
of germinating seeds are used for the formation of the carbohydrates that fuel the growth and development
of the organism.

The glyoxylate cycle can also provide plants with another aspect of metabolic diversity. This cycle allows
plants to take in acetate both as a carbon source and as a source of energy. Acetate is converted to acetyl
CoA (similar to the TCA cycle). This acetyl CoA can proceed through the glyoxylate cycle, and some
succinate is released during the cycle. The four carbon succinate molecule can be transformed into a variety
of carbohydrates through combinations of other metabolic processes; the plant can synthesize molecules
using acetate as a source for carbon. The acetyl CoA can also react with glyoxylate to produce some
NADPH from NADP+, which is used to drive energy synthesis in the form of ATP later in the electron
transport chain.

- Pathogenic fungi
The glyoxylate cycle may serve an entirely different purpose in some species of pathogenic fungi. The
levels of the main enzymes of the glyoxylate cycle, ICL and MS, are greatly increased upon contact with a
human host. Mutants of a particular species of fungi that lacked ICL were also significantly less virulent in
studies with mice compared to the wild type. The exact link between these two observations is still being
explored, but it can be concluded that the glyoxylate cycle is a significant factor in the pathogenesis of
these microbes.

- Vertebrates
Vertebrates were once thought to be unable to perform this cycle because there was no evidence of its two
key enzymes, isocitrate lyase and malate synthase. However, some research suggests that this pathway may
exist in some, if not all, vertebrates. Specifically, some studies show evidence of components of the
glyoxylate cycle existing in significant amounts in the liver tissue of chickens. Data such as these support
the idea that the cycle could theoretically occur in even the most complex vertebrates. Other experiments
have also provided evidence that the cycle is present among certain insect and marine invertebrate species,
as well as strong evidence of the cycle's presence in nematode species. However, other experiments refute
this claim. Some publications conflict on the presence of the cycle in mammals: for example, one paper has
stated that the glyoxylate cycle is active in hibernating bears, but this report was disputed in a later paper.
Evidence exists for malate synthase activity in humans due to a dual functional malate/B-methylmalate

26
synthase of mitochondrial origin called CLYBL expressed in brown fat and kidney. Vitamin D may
regulate this pathway in vertebrates.

Inhibition of the glyoxylate cycle

Due to the central role of the glyoxylate cycle in the metabolism of pathogenic species including fungi and
bacteria, enzymes of the glyoxylate cycle are current inhibition targets for the treatment of diseases. Most
reported inhibitors of the glyoxylate cycle target the first enzyme of the cycle (ICL). Inhibitors were
reported for Candida albicans for potential use as antifungal agents. The mycobacterial glyoxylate cycle is
also being targeted for potential treatments of tuberculosis.

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