Study Questions Week 9: Part I Answers

Pyruvate Dehydrogenase and Citric acid cycle

Part I:
1. (a) What is the reaction catalyzed by the pyruvate dehydrogenase complex (no structures are necessary)?
(b) What is the role of each enzyme.
(c) How is this complex regulated?
(d) Why is this complex regulated?

2. Explain how multienzyme complexes can enhance reaction rates.

3. Explain how multienzyme complexes can “channel” reaction intermediates.

4. Why is Acetyl-CoA an inhibitor of PDH, but an activator of PDH kinase? Include the general activities of the
enzymes, and where acetyl-CoA will function as a regulator on each enzyme.

5. What is meant by the statement that citrate synthase is the pacemaker enzyme of the citric acid cycle?

6. What controls the rate of the reaction catalyzed by citrate synthase?

7. Isocitrate dehydrogenase is a key enzyme in the TCA cycle.

a. Write the reaction catalyzed by this enzyme including structures of the reactants and products (not of any
cofactors).
b. What cellular conditions lead to inhibition of IDH?
c. Explain how inhibition of this enzyme leads to inhibition of glycolysis. Additionally diagram the reactions
involved to illustrate your explanation.

8. What reaction does α-Ketoglutarate dehydrogenase catalyze in the TCA cycle. Why is it important that this
enzyme is regulated? What other enzymes does its product regulate, and why is this necessary? (Do Not include
NADH in your answer).

9. How is α-Ketoglutarate dehydrogenase similar to pyruvate dehydrogenase? How is it different? How might
the enzyme achieve these differences?

10. How is the energy contained in thioester bonds harnessed or utilized within the central metabolic pathway?

11. Provide a rationale for regulating α-Ketoglutarate dehydrogenase. Suggest one positive and one negative
effector of this enzyme (other than an adenine nucleotide).

12. How does succinyl-CoA facilitate substrate level phosphorylation? Discuss all intermediates in the enzymatic
mechanism.

13. Indicate the reaction catalyzed by succinate dehydrogenase. Include structures of reactants and products,
but not cofactors. Briefly indicate how this enzyme and the reaction it catalyzes differs from other oxidation-
reduction reactions (and their enzymes) of the citric acid cycle in terms of location, electron acceptor, and
ultimate production of ATP.
14. The ΔGo’ for the reaction catalyzed by malate dehydrogenase is +29.7 kJ/mol.
a. Indicate how this thermodynamically unfavorable reaction manages to proceed.
b. Suggest how this thermodynamically unfavorable reaction contributes to control of the rate of the citric
acid cycle.

15. Write a balanced equation for the citric acid cycle beginning with acetyl-CoA.
a. Where do the carbons from Acetyl-CoA end up?

16. What is meant by an amphibolic pathway?

17. Identify the reaction catalyzed by pyruvate carboxylase. Discuss the role of this enzyme in the continued
operation of the citric acid cycle and why it is necessary.

18. Briefly indicate the sources of ATP from the citric acid cycle: which reactions (identify by enzyme) give rise to
ATP, or something from which ATP can be formed. Indicate how ATP is produced – a few words to identify the
biological process(es) involved is sufficient.

19. Calculate the net total amount of ATP produced by the complete aerobic catabolism of 1 glucose (indicate
which reaction in the pathway each ATP comes from).

20. What is the total cost (in terms of ATP) of the removal, for biosynthetic pathways, of each of the following
TCA cycle intermediates: (remember to consider the cost of replenishing the intermediates)
a. Citrate
b. α-Ketoglutarate
c. Succinyl-CoA
d. Oxaloacetate

21. You are following the metabolism of pyruvate in which the methyl-C is radioactive: *CH3COCOO-.
Assuming all the pyruvate enters the TCA cycle as acetyl-CoA, indicate the labeling pattern and its distribution
in oxaloacetate first formed by the operation of the TCA cycle.

22. You are following the metabolism of pyruvate in which the carbonyl-C is radioactive: CH3*COCOO-.
Assuming all the pyruvate enters the TCA cycle as acetyl-CoA, indicate the labeling pattern and its distribution
in oxaloacetate first formed by the operation of the TCA cycle.

23. You are investigating the metabolism of glutamic acid in E coli, which enters the TCA cycle as α-Ketoglutarate
following an initial transamination reaction.
a. If you radiolabel the α-carboxyl carbon atom, what fraction of the radioactivity will appear in CO2 during:
i. The conversion of glutamate to oxaloacetate?
ii. The cycling of OAA through the next cycle of the TCA cycle?

b. Indicate the labeling pattern of the following if the side chain carboxyl carbon of glutamate were labeled:
i. Initial OAA:
ii. The α-Ketoglutarate produced in the next turn of the TCA cycle from the OAA labeled in b.i. above.

24. You are investigating the metabolism of the amino acid aspartate in E. coli. After a transamination it enters
the TCA cycle as oxaloacetate. You radiolabel all the carbon atoms of aspartate equally,
a). what fraction of the radioactivity will appear as CO2 during the first 2 turns of the TCA cycle?
b). Draw the structure of OAA and indicate its labeling pattern after the second turn of the TCA cycle.
25. You have a glucose molecule that is radiolabeled at carbon 1.
a). Which carbon(s) in pyruvate would be radiolabeled:

b). Suggest one molecule, other than glycolytic intermediates, before pyruvate that you would be able to detect
radiolabel in. Which carbon would you expect to be labeled?

c). Indicate the pattern and distribution of the label on isocitrate when the pyruvate first
enters the TCA cycle.

d). Indicate the labeling pattern in isocitrate during the next turn of the TCA cycle:

e). What turn of the TCA cycle would label start to be lost as CO2?

*
f). How much of the total label would be lost during the turn in part e

26. You have a glucose molecule that is radiolabeled at carbon 5.

a). Indicate which carbon(s) in pyruvate would be radiolabeled:

b). If pyruvate is converted to oxaloacetate through pyruvate carboxylase, indicate the

labeling pattern on the first isocitrate produced.

c). Indicate the labeling pattern and distribution in oxaloacetate at the end of that turn of the
TCA cycle.:

d). what turn of the TCA cycle would label start to be lost as CO2?
e). Which carbon in pyruvate would have been labeled to give you the labeling pattern in part
c, if that pyruvate went through PDH and the TCA cycle, instead of pyruvate carboxylase.
f). How is the label in part c physically distributed on actual oxaloacetate molecules?

27. Diagram the regulation of the citric acid cycle.

Part II:
28. The following cofactors participate in the reactions catalyzed by the pyruvate dehydrogenase complex.
Identify each cofactor and indicate what part of the molecule participates in catalysis and its role in catalysis.

29. Explain how activity of pyruvate dehydrogenase is controlled when the cell does NOT need to be breaking
down glucose. Consider all the sources of PDH regulators.

30. The citrate produced when acetyl-CoA enters the TCA cycle is asymmetrically converted to isocitrate by the
enzyme aconitase. Briefly describe how the enzyme distinguishes between the two identical substituents on the
central tetrahedral Carbon.

31. What reaction does Isocitrate dehydrogenase catalyze in the TCA cycle? What are the consequences of its
inactivation to both upstream and downstream intermediates?

32. Thioesters are high energy bonds that are found in the central metabolic pathway. Explain how the energy
from the cleavage of these bonds is directly and indirectly used to produce ATP in the TCA cycle.

33. Acetyl-CoA contains a high energy thioester bond.

a). What problem could arise if citrate synthase bound to acetyl-CoA without oxaloacetate?
b). Hexokinase and phosphofructokinase could face the same problem using ATP. How might citrate synthase
avoid the problem in part a?

34. Explain how the rate of the TCA cycle would be affected by the need for its intermediates as precursors for
biosynthetic pathways. Include the difference in the regulation if α-ketoglutarate vs malate is the precursor
removed.
35. You are following the metabolism of pyruvate in which the carbonyl-C is radioactive: CH3*COCOO-.
a. Show the labeling pattern (and its distribution) of the oxaloacetate formed if 50% is produced by the citric
acid cycle and 50% by the pyruvate carboxylase reaction.

b. Indicate the carbon(s) in isocitrate on which label would

be detected during the next turn of the citric acid cycle.

c. If the isocitrate in part b were eventually converted to glutamate,

where would the label be in glutamate?

d. The conversion in part c would remove an intermediate from the TCA cycle. What is the total cost associated
with this removal? SHOW ALL YOUR WORK, Include specific cofactors lost or used.
e. Why does the process in part d occur if it is so costly?
f. If the isocitrate in part b were eventually converted to aspartate, where would the label be in aspartate?

g). What is the total cost of the process in part f? SHOW ALL YOUR WORK, including cofactors lost.

36. You are following the metabolism of pyruvate in which the methyl-C is radioactive: *CH3COCOO-.
a). Show the labeling pattern (and its distribution) of the oxaloacetate formed if 50% is produced by the
citric acid cycle and 50% by the pyruvate carboxylase reaction.

b). Indicate the carbons in isocitrate during the next turn of the citric acid cycle
on which label would be detected.

c). What turn of the TCA cycle would label begin to be lost as CO2? At the end of
that cycle would more label be remaining in the oxaloacetate or be detected in
CO2?