1.  The bottom line. What is the net equation of the citric acid cycle? ✓ 3

2.  A hoax, perhaps? The citric acid cycle is part of aerobic respiration, but no O₂ is required for the cycle. Explain this paradox. ✓ 3

3.  Thing 1 and Thing 2. The citric acid cycle can be thought of as taking place in two stages. What are the two stages? ✓ 3

4.  Like Jack and Jill. Match each enzyme with its description.

5.  One from two. The synthesis of citrate from acetyl CoA and oxaloacetate is a biosynthetic reaction. What is the energy source that drives the formation of citrate? ✓ 3

6.  A penny saved … How is the wasteful hydrolysis of acetyl CoA prevented by citrate synthase? ✓ 3

7.  C₂ + C₂ → C₄.

(a) Which enzymes are required to get the net synthesis of oxaloacetate from acetyl CoA? ✓ 6

(b) Write a balanced equation for the net synthesis.

(c) Do mammalian cells contain the requisite enzymes?

8.  Driving force. What is the value of ΔG°′ for the complete oxidation of the acetyl unit of acetyl CoA by the citric acid cycle? ✓ 4

9.  Acting catalytically. The citric acid cycle itself, which is composed of enzymatically catalyzed steps, can be thought of as essentially a supramolecular enzyme. Explain. ✓ 4

10.  Seven o’clock roadblock. Malonate is a competitive inhibitor of succinate dehydrogenase. How will the concentrations of citric acid cycle intermediates change immediately after the addition of malonate? Why is malonate not a substrate for succinate dehydrogenase? ✓ 4

11.  One of a kind. How is succinate dehydrogenase unique compared with the other enzymes in the citric acid cycle?

12.  Phosphate requirement. What step in the citric acid cycle requires a molecule of inorganic phosphate? ✓ 3

13.  An added bonus. What reaction in the citric acid cycle results in the direct formation of a molecule of ATP? ✓ 3

14.  Regulators. Which enzymes are the key regulatory enzymes of the citric acid cycle itself? ✓ 5

15.  Versatility. What is the chief benefit of being able to perform the glyoxylate cycle?

16.  Kissin’ cousins. How does the decarboxylation of α-ketoglutarate resemble that of pyruvate decarboxylation?

17.  Fats into glucose? Fats are usually metabolized into acetyl CoA and then further processed through the citric acid cycle. In Chapter 17, we learned that glucose could be synthesized from oxaloacetate, a citric acid cycle intermediate. Why, then, after a long bout of exercise depletes our carbohydrate stores, do we need to replenish those stores by eating carbohydrates? Why do we not simply replace them by converting fats into carbohydrates?

18.  No signal, no activity. Why is acetyl CoA an especially appropriate activator for pyruvate carboxylase? ✓ 5

19.  Symmetry problems. In experiments carried out in 1941 to investigate the citric acid cycle, oxaloacetate labeled with ¹⁴C in the carboxyl carbon atom (shown in red below) farthest from the keto group was introduced into an active preparation of mitochondria.



Analysis of the α-ketoglutarate formed showed that none of the radioactive label had been lost. Decarboxylation of α-ketoglutarate then yielded succinate devoid of radioactivity. All of the label was in the released CO₂. Why were the early investigators of the citric acid cycle surprised that all of the label emerged in the CO₂?

20.  Symmetric molecules reacting asymmetrically. The interpretation of the experiments described in problem 19 was that citrate (or any other symmetric compound) cannot be an intermediate in the formation of α-ketoglutarate, because of the asymmetric fate of the label. This view seemed compelling until Alexander Ogston incisively pointed out in 1948 that “it is possible that an asymmetric enzyme which attacks a symmetrical compound can distinguish between its identical groups.” For simplicity, consider a molecule in which two hydrogen atoms, a group X, and a different group Y are bonded to a tetrahedral carbon atom as a model for citrate. Explain how a symmetric molecule can react with an enzyme in an asymmetric way.

23.  Isocitrate lyase and tuberculosis. The bacterium Mycobacterium tuberculosis, the cause of tuberculosis, can invade the lungs and persist in a latent state for years. During this time, the bacteria reside in granulomas—nodular scars containing bacteria and host-cell debris in the center and surrounded by immune cells. The granulomas are lipid-rich, oxygen-poor environments. How these bacteria manage to persist is something of a mystery. Experimental evidence suggests that the glyoxylate cycle is required for the persistence. The following data show the amount of bacteria (presented as colony-forming units, or cfu) in mice lungs in the weeks after an infection.

In graph A, the black data points represent the results for wild-type bacteria and the red data points represent the results for bacteria from which the gene for isocitrate lyase was deleted.



(a) What is the effect of the absence of isocitrate lyase?

The gene encoding isocitrate lyase was reinserted into bacteria from which it had previously been deleted (Chapter 41 describes the reinsertion technique.) In graph B, black data points represent bacteria into which the gene was reinserted and red data points represent bacteria in which the gene was still missing.



[Data for graphs A and B after J. D. McKinney, K. höner zu Bentrup, e. J. Muñozelias, et al., Nature 406:735–738, 2000.]

(b) Do these results support those obtained in part a?

(c) What is the purpose of the experiment in part b?

(d) Why do these bacteria perish in the absence of the glyoxylate cycle?

24.  Flow of carbon atoms. What is the fate of the radioactive label when each of the following compounds is added to a cell extract containing the enzymes and cofactors of the glycolytic pathway, the pyruvate dehydrogenase complex, and the citric acid cycle? (The ¹⁴C label is printed in red.) ✓ 3

(a)  (b)
(c)  (d)

(e) Glucose 6-phosphate labeled at C-1.

25.  Coupling reactions. The oxidation of malate by NAD⁺ to form oxaloacetate is a highly endergonic reaction under standard conditions (ΔG°′ = 29 kJ mol⁻¹, or 7 kcal mol⁻¹). The reaction proceeds readily under physiological conditions.

(a) Why?

(b) Assume an [NAD⁺]/[NADH] ratio of 8 and a pH of 7, and give the lowest [malate]/[oxaloacetate] ratio at which oxaloacetate can be formed from malate.

26.  Power differentials. As we will see in the next chapter, when NADH reacts with oxygen, 2.5 ATP are generated. When FADH₂ reduces oxygen, only 1.5 ATP are generated. Why then does succinate dehydrogenase produce FADH₂ and not NADH when succinate is reduced to fumarate?

27.  Back to Orgo. Before any oxidation can take place in the citric acid cycle, citrate must be isomerized into isocitrate. Why?

28.  Synthesizing α-ketoglutarate. With the use of the reactions and enzymes considered in this chapter and in Chapter 18, pyruvate can be converted into α-ketoglutarate without depleting any of the citric acid cycle components. Write a balanced reaction scheme for this conversion, showing cofactors and identifying the required enzymes. ✓ 6