Pyruvate Dehydrogenase and The Tricarboxylic Acid Cycle
Major Concepts
Respiration is the complete oxidation of organic fuels to CO2 and H2O. Cellular respiration occurs in
three major stages. The first stage oxidizes fuel molecules to the two-carbon molecule acetyl-CoA. In
the second stage (the TCA cycle), acetyl-CoA is oxidized to CO2 and the electron carriers NAD+ and
FAD are reduced. In the third stage (oxidative phosphorylation, discussed later), electrons from the
oxidation of fuel molecules are transferred to O2; ATP is formed as a result of this electron transfer
process.
The first stages of cellular respiration include glycolysis and the production of acetate. Pyruvate, the
product of glycolysis, is converted into acetyl-CoA and CO2 by the pyruvate dehydrogenase
complex. This large enzyme complex consisting of three different enzymes is regulated by allosteric
mechanisms and by covalent modification. This is a good example of substrate channeling by an
enzyme.
The TCA cycle is a series of eight enzyme-catalyzed chemical reactions. For each turn of the cycle,
two carbons enter as an acetyl group and two carbons leave as molecules of
CO2. Oxaloacetate combines with acetyl-CoA to form citrate, which “carries” the carbons from acetylCoA into the cycle. Oxaloacetate is regenerated at the end of the cycle. The citric acid cycle produces
energy for cells in the form of nucleotide triphosphates (ATP or GTP) and reduced electron carriers
FADH2 and NADH.
TCA cycle intermediates also provide precursors for the synthesis of a number of biomolecules.
Intermediates “lost” from the cycle in this way are replenished through a variety of processes called
anaplerotic reactions.
The TCA cycle is under tight regulation. Regulation occurs at the conversion of pyruvate to acetyl-CoA
and at three points in the cycle itself: the entry of acetyl-CoA into the cycle and both of the oxidative
decarboxylation steps. The primary enzymes involved in regulation are the pyruvate dehydrogenase
complex, citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase.
What to Review
Reviewing some of the relevant concepts learned in BB490 should make this chapter more
understandable:
Review the reactions and products of glycolysis and gluconeogenesis. Use this review to also test your
knowledge on redox reactions and how redox chemistry is at the heart of catabolism. In this regard,
also review the roles of FAD and NAD+ as electron carriers. Reduced forms of these nucleotides are
generated in the citric acid cycle and play a critical role in the oxidative phosphorylation of ADP.
Know how prosthetic groups derived often derived from vitamins participate in chemical reactions.
For example, TPP is an important cofactor used by fermentative and citric acid cycle enzymes. Review
how TPP is a carrier of 2-carbon groups and mechanistically how it performs its task.
Review what is meant by reaction equilibria and the relationships among ΔG, ΔG′°, and K'eq. These
concepts are important to an understanding of the regulation of metabolic pathways such as the TCA
cycle.
Topics for Discussion
Answering each of the following questions, especially in the context of a study group discussion, or
during our Zoom sessions should help you understand the important points of the lecture topic.
Production of Acetyl-CoA (Activated Acetate)

The Pyruvate dehydrogenase complex catalyzes a highly exergonic reaction overall. How is the
energy released by this reaction conserved?

The Pyruvate Dehydrogenase Complex employs five coenzymes. Can you name them and list
the function of each?

Make sure you can recognize the structure of coenzyme A; pay attention to the components of
the molecule and to its general function. Why is the thioester bond important to the function of
coenzyme A?

What are the two possible roles of lipoate in enzymatic reactions?
The Pyruvate Dehydrogenase Complex Consists of Three Distinct Enzymes

Be sure you understand how the different enzyme subunits of pyruvate dehydrogenase and
their cofactors act together to catalyze the decarboxylation of pyruvate.

What is the result of the first three reactions of the overall pyruvate dehydrogenase complex
reaction? What is the source of energy for this set of reactions?

Why is it advantageous to have three different enzymatic activities clustered into a single
enzyme complex?
Reactions of the TCA cycle

What are the roles of the TCA cycle? Where in eukaryotes do these reactions take place?

The sequence of reactions in the TCA cycle makes chemical sense. Describe each “stage” of the
cycle, detailing the chemical logic of each.

Why do carbonyl groups “rule” in chemical transformations of metabolic pathways?

Create your own TCA cycle by drawing the structures and names of the intermediates and the
names of the enzymes and cofactors for each step. Alternatively, do the challenge question at
the end of this study guide to follow a labeled carbon through the cycle. Use this exercise to not
only know where the carbon goes in each round of the TCA Cycle, but also to check your
knowledge of each step in the pathway.
Enzymes of the TCA Cycle:
1. Citrate synthase: what is the advantage of having oxaloacetate bind first, which then creates a
binding site for acetyl-CoA?
2. Formation of isocitrate via cis-aconitate: What is the general function of the iron-sulfur center
in aconitase?
3. Oxidation of isocitrate to α-ketoglutarate and CO2: Why is a Mn atom needed for the reaction
to proceed?
4. Oxidation of α-ketoglutarate to succinyl-CoA and CO2: How is the energy of oxidation of αketoglutarate conserved? What are the similarities between the α-ketoglutarate dehydrogenase
complex and the pyruvate dehydrogenase complex? What are the differences?
5. Conversion of Succinyl-CoA to Succinate: What is the source of the energy used to drive the
substrate-level phosphorylation of GDP?
6. Oxidation of succinate to fumarate: Explain why it is significant that the electron carrier FAD is
covalently bound to succinate dehydrogenase, whereas NAD+ is in a soluble, unconjugated
form.
7. Hydration of fumarate to malate: This reaction is readily reversible, with a ΔG′° of −3.8
kJ/mol. Why do you think it proceeds in the direction of malate formation in vivo?
8. Malate conversion to oxaloacetate: How can malate dehydrogenase operate to produce
oxaloacetate when the isolated enzyme clearly operates to reduce oxaloacetate to malate?
Further test your understanding of the TCA cycle

Which reaction(s) of the TCA cycle store(s) the energy derived from oxidations as NADH? Which
store(s) it as FADH2?
 Why is the oxidation of acetate so complicated?

What does the term “amphibolic” mean? The term “anapleurotic”? Test yourself further by
giving examples of the main exit and entry points for metabolites leaving and entering the
cycle.
Regulation of the TCA Cycle
 What do all the allosteric activators of the pyruvate dehydrogenase complex signal about the
cell’s energy state? What do the allosteric inhibitors signal?
 How does covalent modification regulate the pyruvate dehydrogenase complex in mammals?
 Why are citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase good
candidates for regulatory enzymes?
SELF-TEST
Do You Know the Terms?




The metabolic block of enzyme reactions that require energy input and reducing power for
synthesis reactions.
The glyoxylate cycle occurs in these organelles.
Describes pathways used in both anabolism and catabolism; for example, the TCA cycle, in
which oxaloacetate is an intermediate in the degradation and the synthesis of glucose.
Cofactor involved in the decarboxylation of pyruvate and the initial binding of the resulting
hydroxy-ethyl group to pyruvate dehydrogenase.





General type of reaction catalyzed by isocitrate dehydrogenase and by α-ketoglutarate
dehydrogenase complex. (2 words)
Acyl groups are linked to coenzyme A through a _____ bond and are thus activated for group
transfer.
Consuming too much eggnog or too many raw eggs may cause a deficiency in the vitamin
_____, which is required as a cofactor for pyruvate carboxylase.
Compound formed by the hydration of fumarate.
Pyruvate dehydrogenase cofactor involved in the transfer of acyl groups and of electrons.
Practice Questions

The underlined portion of the following statements about the PDH Complex IS CORRECT, but
the rest of the statement contains one or more obvious errors. Fix the error(s) by crossing out
the false portion of the statement and re-write it so that the entire statement reads true:
A. The products of the overall PDH Complex are ATP, FAD, and succinyl-CoA
B. Three cofactors that are bound to proteins of the PDH Complex, which also participate in
catalysis are riboflavin, thiamine, biotin.
C. Lipoic acid acts as part of a long flexible arm to move activated acetyl-groups from E1 to E3
D. In mammals, a high [NADH]/[NAD+] ratio would cause a pyruvate dehydrogenase
phosphatase to become active, which in turn would activate the PDH Complex.

Which of the following correctly describes the TCA cycle?
A.
B.
C.
D.
E.
Oxygen is required to regenerate electron acceptors.
Citrate is dehydrated and hydrated by the same enzyme, aconitase.
ATP is formed in three of the TCA reactions
Succinate dehydrogenase links the TCA cycle to the electron transport chain.
All of the above are true.

All of the following enzymes are linked to the reduction of NADH except:
A. isocitrate dehydrogenase.
B. lactate dehydrogenase.
C. succinate dehydrogenase.
D. pyruvate dehydrogenase.
E. α-ketoglutarate dehydrogenase.

In questions (A-G which are listed below the potential answers), match the descriptions to
the appropriate enzyme(s) from the list below. (There can be more than one answer to a
question.)
1.
Citrate synthase
2.
3.
4.
5.
6.
Isocitrate dehydrogenase
Pyruvate dehydrogenase
Succinate dehydrogenase
Succinyl-CoA synthetase
α-Ketoglutarate dehydrogenase
__2,3,6__ A. Allosterically activated by calcium.
__1__ B. Catalyzes the committed step in the TCA cycle.
__4__ C. The only membrane-bound enzyme in the TCA cycle.
__5__ D. Catalyzes the substrate-level phosphorylation of ADP or GDP.
___3_ E. Regulated by reversible phosphorylation.
__2,3,6__ F. Inhibited by NADH.
__1,2__ G. Activated by AMP or ADP.
Challenge Question
•
White circles denote carbons of the TCA Cycle intermediates
•
The Black circle is the carbonyl-carbon of an incoming acetyl-CoA molecule. It is labelled with a
fluorescent compound so that it can be followed enters the TCA Cycle
1. Which carbon(s) are labelled after 1 turn (ie to oxaloacetate)?
2. Two turns?