Yuan Provido

Chem 2065 AY2023-24

1. Define metabolism, catabolism, and anabolism (amphibolic, Krebs cycle, tricarboxylic acid cycle)
- Intro to Metabolism
o In the cell, complex substances are broken down for energy, metabolites,
structural components, etc.
o Cells must synthesize new complex substances
o Thousands of such reactions are occurring simultaneously in a single cell
o These reactions occur with a minimum of side products, energy loss or
undesired interferences and at reasonable temperatures, pH and pressure
o All these reactions controlled or regulated for optimum efficiency
- Types of Metabolism
o Catabolism
▪ Breakdown of complex substances / oxidative breakdown of nutrients
▪ Energy Yielding process

▪ Higher energy to Lower energy

▪ (Spontaneous)
o Anabolism
▪ Synthesis of complex substances from simpler ones / reductive
synthesis of biomolecules
▪ Energy Requiring process

▪ Lower energy to Higher Energy

▪ (Non-spontaneous)
- The Citric Acid Cycle, AKA, The Krebs cycle, AKA the tricarboxylic acid cycle
o A central metabolic pathway and is apart of aerobic metabolism
o Is an amphibolic process wherein it plays a role in both the
anabolism and catabolism processes.
▪ Although this process is a part of the pathway of aerobic oxidation of
nutrients which is a catabolic pathway, some of the molecules that are
included in this cycle are the starting points of biosynthetic pathways
which are anabolic
o It is called the ‘Krebs Cycle’ because it was named after the person who first
investigated this particular pathway – the name of the person is Sir Hans Krebs.
o It is called the ‘Tricarboxylic acid cycle’ due to the involvement of some molecules
with particular acids containing three carboxyl group
 o
▪ Pyruvate is oxidized and acetate is transferred to CoA (converted into a 2-
carbon group)
▪ Acetyl CoA enters the TCA cycle, acetate is oxidized to CO2
2. Provide an overview of the mitochondrial structure (matrix, cristae, inner
membrane, intermembrane space)
- Mitochondrial matrix
o The part of the mitochondrion enclosed by the inner mitochondrial membrane
o The reactions of the citric acid cycle take place in the matrix, except for the
one in which the intermediate electron acceptor is FAD
o The decarboxylation of pyruvate, and the oxidation of acetate takes place
in the mitochondrial matrix
- Intermembrane space
o the region between the inner and outer mitochondrial membranes
- Inner membrane
o A tight barrier between the matrix and the cytosol, and very few
compounds can cross this barrier without a specific transport protein
- Cristae
o The folds in between the outer membrane and the inner membrane
 -
3. Provide an overview of the reactions in the citric acid cycle (NADH, FADH(sub2), oxidative
decarboxylation)
- In the first reaction of the cycle, the two-carbon acetyl group condenses with the
four- carbon oxaloacetate ion to produce the six-carbon citrate ion. In the next
few steps, the citrate isomerizes, and then it both loses carbon dioxide and is
oxidized.
o This process, called oxidative decarboxylation, produces the five carbon
compound - ketoglutarate, which again is oxidatively decarboxylated to
produce the four-carbon compound succinate. The cycle is completed by
regeneration of oxaloacetate from succinate in several steps.
▪ Oxidative Decarboxylation
• Net Reaction: Pyruvate -> Acetyl-CoA (side product is CO2)
• Three-step reaction involving the pyruvate dehydrogenase complex
▪ Complex – arrangement of three different sets
of enzymes
st
o 1 enzyme- Dihydrolipoyl transacetylase (TA); 24 units
 o 2nd enzyme – Pyruvate dehydrogenase (PDH); 12
dimeric units

o 3rd enzyme– Dihydrolipoyl dehydrogenase (DLD) ; 12 units

- The citric acid cycle has eight steps, each catalyzed by a different enzyme. Four of
the eight steps—Steps 3, 4, 6, and 8—are oxidation reactions (see Figure 19.3). The
oxidizing agent is NAD1 in all except Step 6, in which FAD plays the same role. In
Step 5, a molecule of GDP (guanosine diphosphate) is phosphorylated to produce
GTP. This reaction is equivalent to the production of ATP because the phosphate
group is then easily transferred to ADP, producing GDP and ATP.
- Logic of TCA cycle
o Cleavage of 2-carbon compound
▪ 2-C fragment -> 2 CO2 (need)
• CH3COOH -> 2 CO2 (either acetic acid or acetyl group)
o Organic reactions
 ▪ Reverse of aldol condensation (Beta-Cleavage):
• Cleavage of C(beta) -C(alpha)-C=O

• Example: Fructose bisphosphate aldolase reaction in glycolysis

▪ Cleavage of alpha-hydroxyketone

o Neither reaction is suitable for acetate

o TCA Cycle solution: condenses acetate with oxaloacetate then carry out
Beta- cleavage
4. Explain the individual roles of the different enzymes (pyruvate dehydrogenase,
dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase, pyruvate dehydrogenase
kinase, and pyruvate dehydrogenase phosphatase) convert pyruvate into acetyl-CoA
(TPP, hydroxyethyl TPP, lipoic acid) Note: This is a fascinating reaction as well as the
architecture of the whole enzyme complex
-
- The pyruvate dehydrogenase complex is a multienzyme complex that catalyzes the
conversion of pyruvate to acetyl-CoA and carbon dioxide.
- In mammals, five enzymes make up the pyruvate dehydrogenase complex
o Essentially, the first three are involved in the conversion of pyruvate to
acetyl-CoA. The kinase and the phosphatase are enzymes used in the control
of PDH and are present on a single polypeptide. The reaction takes place in
five steps. Two enzymes catalyze reactions of lipoic acid, a compound that
has a disulfide group in its oxidized form and two sulfhydryl groups in its
reduced form.
▪ Lipoic acid can act simply as an oxidizing agent, or it can
simultaneously take part in two reactions—a redox reaction and the
shift of an acetyl group by transesterification
o pyruvate dehydrogenase
▪ The first step in the reaction sequence that converts pyruvate to
carbon dioxide and acetyl-CoA is catalyzed by pyruvate
dehydrogenase
▪ This enzyme requires thiamine pyrophosphate (TPP; a metabolite of
vitamin B1, or thiamine) as a coenzyme. The coenzyme is not
covalently bonded to the enzyme; they are held together by
noncovalent interactions. Mg2+ is also required.
▪ In the pyruvate dehydrogenase reaction, an -keto acid, pyruvate, loses
carbon dioxide; the remaining two-carbon unit becomes covalently
bonded to TPP
o dihydrolipoyl transacetylase
▪ The second step of the reaction is catalyzed by dihydrolipoyl
transacetylase. This enzyme requires lipoic acid as a coenzyme. The
lipoic acid is covalently bonded to the enzyme by an amide bond to
the ´-amino group of a lysine side chain. The two-carbon unit that
originally came from pyruvate is
 transferred from the thiamine pyrophosphate to the lipoic acid, and, in
the process, a hydroxyl group is oxidized to produce an acetyl group.
Lastly, the acetyl group is now covalently bonded to the lipoic acid by
a thioester linkage
▪ The third step of the reaction is also catalyzed by dihydrolipoyl
transacetylase. A molecule of CoA-SH attacks the thioester linkage,
and the acetyl group is transferred to it. The acetyl group remains
bound in a thioester linkage
▪ The reduced form of lipoic acid remains covalently bound to
dihydrolipoyl transacetylase. The reaction of pyruvate and CoA-SH
has now reached the stage of the products, carbon dioxide and
acetyl-CoA, but the lipoic acid coenzyme is in a reduced form
o dihydrolipoyl dehydrogenase
▪ In the fourth step of the overall reaction, the enzyme dihydrolipoyl
dehydrogenase reoxidizes the reduced lipoic acid from the sulfhydryl
to the disulfide form. The lipoic acid still remains covalently bonded
to the transacetylase enzyme. The dehydrogenase also has a
coenzyme, FAD that is bound to the enzyme by noncovalent
interactions. As a result, FAD is reduced to FADH2. FADH2 is
reoxidized in turn. The oxidizing agent is NAD1, and NADH is the
product along with reoxidized FAD. Enzymes such as pyruvate
dehydrogenase are called flavoproteins because of their attached
FADs.
o pyruvate dehydrogenase kinase
o pyruvate dehydrogenase phosphatase
o Enzymes and Coenzymes
▪ Pyruvate + CoA + NAD+ -> Acetyl CoA + CO2 + NADH + H+
▪ The complex uses five coenzymes
• Three are prosthetic groups which means that these are
covalently bound to their enzymes
o TPP (thiamine pyrophosphate)

o Lipoamide
 oFAD/FADH2
• Two are transiently associated with the complex
o CoA
o NAD+/NADH
o Reaction Mechanism

• 1st step: Pyruvate loses CO2 and Hydroxyethyl TPP is formed

o Pyruvate interacts with Thiamine pyrophosphate, its
carbonyl group is oxidized, while the alcohol group is
reduced (AKA the acetyl group connects with the
enzyme, is reduced)
o Enzyme that is being exhibited in this step is
pyruvate dehydrogenase – redox reaction
• nd
2 Step: Hydroxyethyl group is transferred to lipoic
acid and oxidized to form acetyl dihydrolipoamide
o Enzyme that is being exhibited in this step is
Dihydrolipoyl transacetylase – redox reaction
 o Lipoic acid (oxidized form due to its disulfide linkage)
which is a part of the protein, swings around, attaches
itself to where the TPP is located in the pyruvate
dehydrogenase complex and it picks up the
hydroxyethyl group and oxidizes it back into the
carbonyl group
• 3rd step: Acetyl group is transferred to CoA
o CoASH – SH is thiol group / thioster
o Lipoic acid is fully reduced, but in this form – it is useless
o Dihydrolipoamide is reoxidized by FAD, which is
oxidized by NAD+
▪ Once Dihydrolipoamide is reoxidized by FAD to
form the lipoic acid
o Enzyme that is being exhibited in this step is
Dihydrolipoyl dehydrogenase

o Role of TPP
▪ The coenzyme form of the vitamin (B-1) thiamine that functions
in the decarboxylation of alpha-ketoacids.

5. Write the eight reactions of the citric acid cycle including the enzymes involved

-
- Step 1

o
- Step 2

o
- Step 3

o
- Step 4

o
- Step 5
 o
- Step 6

o
- Step 7

o
- Step 8

o
6. Explain the chemical logic behind the formation of citric acid
 - This reaction is called a condensation because a new carbon– carbon bond is
formed. The condensation reaction of acetyl-CoA and oxaloacetate to form citryl-
CoA takes place in the first stage of the reaction
o The reaction is catalyzed by the enzyme citrate synthase, originally called
“condensing enzyme.” A synthase is an enzyme that makes a new covalent
bond during the reaction, but it does not require the direct input of ATP
▪ Nucleophilic attack by alpha-carbon of carbonyl carbon of oxaloacetate
▪ Thus producing Citryl-CoA as an intermediate
▪ This Citryl-CoA is easily hydrolyzed into Citrate
▪ Highly exergonic, spontaneous reaction
▪ This reaction is irreversible, considered as a control step in the
overall mechanism

▪ Allosteric inhibitors: NADH, succinyl-CoA (from step 5 in TCA cycle)

7. Explain the chemical logic behind the conversion of citrate into isocitrate
- One of the most interesting features of the reaction is that citrate, a symmetrical
(achiral) compound, is converted to isocitrate, a chiral compound, a molecule that
cannot be superimposed on its mirror image.
o It is often possible for a chiral compound to have several different
isomers. Isocitrate has four possible isomers, but only one of the four is
produced by this reaction
- Aconitase, the enzyme that catalyzes the conversion of citrate to isocitrate, can
select one end of the citrate molecule in preference to the other.
o This type of behavior means that the enzyme can bind a symmetrical substrate
in an unsymmetrical binding site.
o The enzyme forms an unsymmetrical three-point attachment to the citrate molecule

o
▪ Iron coordinates C-3 carboxyl and hydroxyl of citrate
▪ Equilibrium favors citrate (higher concentrations of citrate is needed to
push the reaction forward)
- Citrate, which contains tertiary alcohol, is a poor candidate for further oxidation
o Solution: isomerization to a secondary alcohol
 o Enzyme is stereospecific: Removes only the pro-RH atom of the pro-R arm of citrate
8. Explain why the formation of alpha-ketoglutarate is a control point in the citric acid cycle
- This reaction is the first of two oxidative decarboxylations of the citric acid cycle;
- This is the first of the reactions in which NADH is produced. One molecule of
NADH is produced from NAD1 at this stage by the loss of two electrons in the
oxidation. As we saw in our discussion of the pyruvate dehydrogenase complex, each
NADH produced leads to the production of 2.5 ATP in later stages of aerobic
metabolism. Recall also that there will be two NADH, equivalent to five ATP, for
each original molecule of glucose.

o Redox reaction
o First oxidative decarboxylation step in the TCA cycle
o Secondary alcohol is oxidized to ketone while NAD+ is reduced to NADH
o Followed by Beta-decarboxylation step
▪ Inhibited by ATP and NADH
▪ Activated by ADP
9. Explain why the formation of succinyl-CoA is a control point in the citric acid cycle
- The second oxidative decarboxylation takes place in Step 4 of the citric acid cycle
o At this point, two molecules of CO2 have been produced by the oxidative
decarboxylations of the citric acid cycle. Removal of the CO2 makes the
citric acid cycle irreversible in vivo, although in vitro each separate
reaction is reversible
10. Compare the previous reaction with the reaction that converts pyruvate to acetyl-CoA
- This reaction is similar to the one in which acetyl-CoA is formed from pyruvate,
with NADH produced from NAD1. Once again, each NADH eventually gives rise to
2.5 ATP, with five ATP from each original molecule of glucose.
 - The reaction occurs in several stages and is catalyzed by an enzyme system called the
Alpha- ketoglutarate dehydrogenase complex, which is very similar to the pyruvate
dehydrogenase complex. Each of these multienzyme systems consists of three
enzymes that catalyze the overall reaction. The reaction takes place in several steps,
and there is again a requirement for thiamine pyrophosphate (TPP), FAD, lipoic acid,
and Mg+2. This reaction is highly exergonic as is the one catalyzed by pyruvate
dehydrogenase
11. Explain the chemical logic behind the conversion of succinyl-CoA into succinate
- This reaction step is called substrate-level phosphorylation to distinguish it from the
type of reaction for production of ATP that is coupled to the electron transport chain
o In substrate-level phosphorylation, the free energy of hydrolysis of the
other compound (succinyl-CoA here) provides the energy for the
phosphorylation reaction. The production of ATP in this reaction is the
only place in the citric acid cycle in which chemical energy in the form of
ATP is made available to the cell.

, Close to equilibrium – not a

control point
▪ GTP is energetically equivalent to ATP
• GTP + ADP -><- GDP + ATP
• Nucleoside diphosphokinase
o Transfers the phosphate group – from gtp into adp to
form atp
12. Explain the chemical logic behind the conversion of succinate into fumarate
- The electron acceptor, which is FAD rather than NAD1, is covalently bonded to the
enzyme; succinate dehydrogenase is also called a flavoprotein because of the
presence of FAD with its flavin moiety. In the succinate dehydrogenase reaction,
FAD is reduced to FADH2 and succinate is oxidized to fumarate.

o
o The E-FAD and E-FADH2 in the equation indicate that the electron acceptor is
covalently bonded to the enzyme. The FADH2 group passes electrons on to
the electron transport chain, and eventually to oxygen, and gives rise to 1.5
ATP, rather than 2.5, as is the case with NADH
 ▪ total equilibrium
13. Explain the chemical logic behind the conversion of fumarate into
malate
- Water is added across the double bond of fumarate in a hydration reaction to give malate
o Trans-Hydration of fumarate to form L-malate

14. Explain the chemical logic behind the conversion of malate into
oxaloacetate
- Malate is oxidized to oxaloacetate indicating that the cycle is completed
o The oxaloacetate can then react with another molecule of acetyl-CoA to
start another round of the cycle

o
o Highly positive, driving force of reaction is removal of products =
15. Explain how each reaction is regulated to either go in the forward or the reverse
direction (energetics or equilibrium?)

-
- Regulation of the TCA cycle
o Three sites in TCA cycle are key sites for regulation
▪ Step 1: Citrate synthase – ATP, NADH and succinyl-CoA inhibit
▪ Step 3: Isocitrate dehydrogenase – ATP inhibits, ADP and NAD+ activate
▪ Step 4: Alpha-Ketoglutarate dehydrogenase – NADH and
succinyl-CoA inhibit, AMP activates
 o Common theme
▪ ATP, NADH, acetyl-CoA are indicators that the cell has high energy,
thus the TCA must be inhibited
▪ ADP, NAD+, AMP are indicators that the cell has low energy,
especially if these are high in concentration thus TCA must be
activated
o Pyruvate dehydrogenase is a highly regulated enzyme
▪ ATP, NADH, acetyl-CoA inhibitors

16. Explain why plants are able to produce carbohydrates from acetyl-CoA whereas animals can’t
(glyoxylate pathway)
- Two enzymes are responsible for the ability of plants and bacteria to produce
glucose from fatty acids. Isocitrate lyase cleaves isocitrate, producing glyoxylate
and succinate while Malate synthase catalyzes the reaction of glyoxylate with
acetyl-CoA to produce malate
- These two reactions in succession bypass the two oxidative decarboxylation steps
of the citric acid cycle. The net result is an alternative pathway, the glyoxylate
cycle
o glyoxylate cycle is a pathway in plants that is an alternative to the citric
acid cycle and that bypasses several citric acid cycle reactions
▪ This pathway results in the net conversion of two acetyl-
CoA to oxaloacetate.
- Glyoxylate Cycle
o Using Acetate as Sole Carbon Source (Exclusive to Plants)
▪ Using the intermediates of TCA cycle for anabolism will
deplete such intermediates
 ▪ Thus animals cannot use acetate or other compounds that are
converted to acetate alone, as a sole carbon source
▪ However plants (seedlings) and some bacteria and algae have
enzymes that can short circuit the CAC

17. Explain the role of the citric acid cycle in catabolism (breakdown of biomolecules
aside from glucose such as amino acids and lipids)
- Proteins, carbohydrates, and triacyglycerols can be broken down into amino acids,
sugar monomers, and acetyl-CoA respectively; molecules that can all enter into
the citric acid cycle.

-
18. Explain the role of the citric acid cycle in anabolism (formation of biomolecules such as glucose,
amino acids, lipids, and nucleic acids)
- The malate reaction is an important source of NADPH for lipid anabolism, with the
pentose phosphate pathway the only other source.
- Anaplerotic Reactions
o Reactions that replenish TCA cycle intermediates
o These are needed in order to sustain gluconeogenesis
▪ PEP carboxylase – converts PEP to oxaloacetate
▪ Pyruvate carboxylase – converts pyruvate to oxaloacetate
▪ Malic enzyme converts pyruvate into malate
 ▪
o PEP carboxykinase – could have been an anaplerotic reaction but it goes the wrong
way
o CO2 binds weakly to the enzyme, but oxaloacetate binds tightly, so the reaction
goes the “wrong” way

-
19. [Assignment: Differentiate between glucogenic and ketogenic amino acids]
- Glucogenic Amino Acids - can be converted into glucose through gluconeogenesis.
o Intermediate product: Pyruvate and other glucose precursors
o Implications towards TCA
▪ Enter TCA as pyruvate or as TCA intermediates
▪ If intermediates are used up and not get depleted, then
gluconeogenesis is sustained
- Ketogenic Amino Acids - can be converted into ketone bodies
o Intermediate product: Acetyl CoA and acetoacetyl CoA
o Implications towards TCA
▪ Enter TCA only as Acetyl-CoA
▪ Cannot rely on using the intermediates of the citric acid cycle to
undergo gluconeogenesis