A one-way link. What reaction serves to link glycolysis and the citric acid cycle, and what is the enzyme that catalyzes the reaction?

Naming names. What are the five enzymes (including regulatory enzymes) that constitute the pyruvate dehydrogenase complex? Which reactions do they catalyze?

Coenzymes. What coenzymes are required by the pyruvate dehydrogenase complex? What are their roles?

More coenzymes. Distinguish between catalytic coenzymes and stoichiometric coenzymes in the pyruvate dehydrogenase complex.

Waste and fraud? Figure 17.9 shows the steps in the pyruvate dehydrogenase complex reaction cycle. A key product, acetyl CoA, is released after the fourth step. What is the purpose of the remaining steps?

Joined at the hip. List some of the advantages of organizing the enzymes that catalyze the formation of acetyl CoA from pyruvate into a single large complex.

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 citric acid cycle, and the pyruvate dehydrogenase complex? (The ¹⁴C label is printed in red.)

C₂ + C₂ → C₄

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

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

A potent inhibitor. Thiamine thiazolone pyrophosphate binds to pyruvate dehydrogenase about 20,000 times as strongly as does thiamine pyrophosphate, and it competitively inhibits the enzyme. Why?

Like Holmes and Watson. Match each term with its description.

Lactic acidosis. Patients in shock often suffer from lactic acidosis owing to a deficiency of O₂. Why does a lack of O₂ lead to lactic acid accumulation? One treatment for shock is to administer dichloroacetate (DCA), which inhibits the kinase associated with the pyruvate dehydrogenase complex. What is the biochemical rationale for this treatment?

DCA again. Patients with pyruvate dehydrogenase deficiency show high levels of lactic acid in the blood. However, in some cases, treatment with dichloroacetate (DCA) lowers lactic acid levels.

Energy rich. What are the thioesters in the reaction catalyzed by PDH complex?

Alternative fates. Compare the regulation of the pyruvate dehydrogenase complex in muscle and in liver.

Mutations.

Flaking paint, green wallpaper. Clare Boothe Luce, ambassador to Italy in the 1950s (and Connecticut congressperson, playwright, editor of Vanity Fair, and the wife of Henry Luce, founder of Time magazine and Sports Illustrated) became ill when she was staying at the ambassadorial residence in Rome. The paint on the dining room ceiling, an arsenic-based paint, was flaking; the wallpaper of her bedroom in the ambassadorial residence was colored a mellow green owing to the presence of cupric arsenite in the pigment. Suggest a possible cause of Ambassador Luce’s illness.

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

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

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

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

Synthesizing α-ketoglutarate. It is possible, with the use of the reactions and enzymes considered in this chapter, to convert pyruvate 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.

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?

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

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?

Back to Orgo (or do you say O chem?). Before any oxidation can occur in the citric acid cycle, citrate must be isomerized into isocitrate. Why is this the case?

A nod is as good as a wink to a blind horse. Explain why a GTP molecule, or another nucleoside triphosphate, is energetically equivalent to an ATP molecule in metabolism.

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

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

Fats into glucose? Fats are usually metabolized into acetyl CoA and then further processed through the citric acid cycle. In Chapter 16, we saw that glucose can 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?

Alternative fuels. As we will see (Chapter 22), fatty acid breakdown generates a large amount of acetyl CoA. What will be the effect of fatty acid breakdown on pyruvate dehydrogenase complex activity? On glycolysis?

Theme and variation. Propose a reaction mechanism for the condensation of acetyl CoA and glyoxylate in the glyoxylate cycle of plants and bacteria.

Symmetry problems. In experiments carried out in 1941 to investigate the citric acid cycle, oxaloacetate labeled with ¹⁴C in the carboxyl carbon atom farthest from the keto group was introduced to 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 the label was in the released CO₂. Why were the early investigators of the citric acid cycle surprised that all the label emerged in the CO₂?

Symmetric molecules reacting asymmetrically. The interpretation of the experiments described in Problem 34 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 [italics added].” 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.

A little goes a long way. As will become clearer in Chapter 18, the activity of the citric acid cycle can be monitored by measuring the amount of O₂ consumed. The greater the rate of O₂ consumption, the faster the rate of the cycle. Hans Krebs used this assay to investigate the cycle in 1937. He used as his experimental system minced pigeon-breast muscle, which is rich in mitochondria. In one set of experiments, Krebs measured the O₂ consumption in the presence of carbohydrate only and in the presence of carbohydrate and citrate. The results are shown in the following table.

Effect of citrate on oxygen consumption by minced pigeon-breast muscle

Arsenite poisoning. The effect of arsenite on the experimental system of Problem 36 was then examined. Experimental data (not presented here) showed that the amount of citrate present did not change in the course of the experiment in the absence of arsenite. However, if arsenite was added to the system, different results were obtained, as shown in the following table.

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. The results of recent research suggest that the glyoxylate cycle is required for the persistence. The following data show the amount of bacteria [presented as colony-forming units (cfu)] in mice lungs in the weeks after an infection.

522

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