Vocabulary Amphibolic: able to be a part of both anabolism and catabolism Anaplerotic: referring to a reaction that ensures an adequate supply of an important metabolite Citrate Synthase: the enzyme that catalyzes the first step of the citric acid cycle Citric Acid Cycle: a central metabolic pathway; part of aerobic metabolism Electron Transport to Oxygen: a series of oxidation reduction reactions by which the electrons derived from oxidation of nutrients are passed to oxygen Glyoxylate Cycle: a pathway in plants that is an alternative to the citric acid cycle and that bypasses several citric acid cycle reactions Glyoxysomes: membrane-enclosed organelles that contain the enzymes of the glyoxylate cycle Intermembrane Space: the region between the inner and outer mitochondrial membranes A-Ketoglutarate Dehydrogenase Complex: one of the enzymes of the citric acid cycle; it catalyzes the conversion of a-ketoglutarate to succinyl-coa Krebs Cycle: an alternative name for the citric acid cycle Matrix (Mitochondrial): the part of a mitochondrion enclosed within the inner mitochondrial membrane Non-Heme (Iron Sulfur) Protein: a protein that contains iron and sulfur but no heme group Oxidative Decarboxylation: loss of carbon dioxide accompanied by oxidation Oxidative Phosphorylation: a process for generating ATP; it depends on the creation of a ph gradient within the mitochondrion as a result of electron transport Pyruvate Dehydrogenase Complex: a multi-enzyme complex that catalyzes the conversion of pyruvate to acetyl-coa and carbon dioxide Tricarboxylic Acid Cycle: another name for the citric acid cycle Thioester: a sulfur-containing analogue of an ester
Chapter Summary The central role of the citric acid cycle in metabolism The citric acid cycle plays a central role in metabolism. It is the first part of aerobic metabolism; it is also amphibolic (both catabolic and anabolic). Where does the citric acid cycle take place in the cell? Unlike glycolysis, which takes place in the cytosol, the citric acid cycle occurs in mitochondria. Most of the enzymes of the citric acid cycle are in the mitochondrial matrix. Succinate dehydrogenase, the sole exception, is localized in the inner mitochondrial membrane. What are the key features of the citric acid cycle? Pyruvate produced by glycolysis is transformed by oxidative decarboxylation into acetyl- CoA in the presence of coenzyme A. Acetyl-CoA then enters the citric acid cycle by reacting with oxaloacetate to produce citrate. The reactions of the citric acid cycle include two other oxidative decarboxylations, which transform the six-carbon compound citrate into the fourcarbon compound succinate. The cycle is completed by regeneration of oxaloacetate from succinate in a multistep process that includes two other oxidation reactions. The overall reaction, starting with pyruvate, is NAD+ and FAD are the electron acceptors in the oxidation reactions. The cycle is strongly exergonic. How many enzymes are needed to convert pyruvate to acetyl- CoA? Pyruvate is produced by glycolysis in the cytosol of the cell. The citric acid cycle takes place in the matrix of the mitochondria, so the pyruvate must first pass through a transporter into this organelle. There, pyruvate will find pyruvate dehydrogenase, a large, multi-subunit protein made up of three enzymes involved in the production of acetyl-coa plus two enzyme activities involved in control of the enzymes. The reaction requires several cofactors, including FAD, lipoic acid, and TPP.
The individual reactions of the citric acid cycle. Acetyl-CoA condenses with oxaloacetate to give citrate, a six-carbon compound. Citrate isomerizes to isocitrate, which then undergoes an oxidative decarboxylation to a-ketoglutarate, a five-carbon compound. This then undergoes another oxidative decarboxylation producing succinyl-coa, a four-carbon compound. The two decarboxylation steps also produce NADH. Succinyl-CoA is converted to succinate with the concomitant production of GTP. Succinate is oxidized to fumarate, and FADH2 is produced. Fumarate is converted to malate, which is then oxidized to oxaloacetate while another NADH is produced. The overall pathway has a DG ' of 277.7 kj mol21. During the course of the cycle, starting from pyruvate, four NADH molecules and one FADH2 are produced. Between the GTP formed directly and the reoxidation of the reduced electron carriers by the electron transport chain, the citric acid cycle produces 25 ATP. Control of the citric acid cycle is exercised at three points. How does the pyruvate dehydrogenase reaction control the citric acid cycle? There is a control point outside the cycle, the reaction in which pyruvate produces acetyl- CoA. How is control exerted within the citric acid cycle? Within the citric acid cycle, the three control points are the reactions catalyzed by citrate synthase, isocitrate dehydrogenase, and the a-ketoglutarate dehydrogenase complex. In general, ATP and NADH are inhibitors, and ADP and NAD+ are activators of the enzymes at the control points. In plants and bacteria, there is a pathway related to the citric acid cycle: the glyoxylate cycle. The two oxidative decarboxylations of the citric acid cycle are bypassed. This pathway plays a role in the ability of plants to convert acetyl-coa to carbohydrates, a process that does not occur in animals. Like a giant traffic c circle of life, the citric acid cycle has many routes entering it. Many members of the three basic nutrient types proteins, fats, and carbohydrates are metabolized to smaller molecules that can cross the mitochondrial membrane and enter the citric acid cycle as one of the intermediate molecules. In this way, the cycle allows us to get energy from the food we eat. Carbohydrates and many amino acids can enter the cycle either as pyruvate or as acetyl- CoA. Lipids enter as acetyl-coa. Because of the transamination reaction possible with glutamate and a-ketoglutarate, almost any amino acid can be transaminated to glutamate, producing a-ketoglutarate that can enter the cycle. Several other pathways lead to amino acids entering the pathway as succinate, fumarate, or malate. While the citric acid cycle
takes place in mitochondria, many anabolic reactions take place in the cytosol. Oxaloacetate, the starting material for gluconeogenesis, is a component of the citric acid cycle. Malate, but not oxaloacetate, can be transported across the mitochondrial membrane. After malate from mitochondria is carried to the cytosol, it can be converted to oxaloacetate by malate dehydrogenase, an enzyme that requires NAD+. Malate, which crosses the mitochondrial membrane, plays a role in lipid anabolism, in a reaction in which malate is oxidatively decarboxylated to pyruvate by an enzyme that requires NADP1, producing NADPH. How is lipid anabolism related to the citric acid cycle? The malate reaction is an important source of NADPH for lipid anabolism, with the pentose phosphate pathway the only other source. How is amino acid metabolism related to the citric acid cycle? In addition, most of the intermediates have anabolic pathways leading to amino acids and fatty acids, as well as some that lead to porphyrins or pyrimidines.
Questions and Answers 1. Which pathways are involved in the anaerobic metabolism of glucose? Which pathways are involved in the aerobic metabolism of glucose? Anaerobic glycolysis is the principal pathway for the anaerobic metabolism of glucose. The pentose phosphate pathway can also be considered. Aerobic glycolysis and the citric acid cycle are responsible for the aerobic metabolism of glucose. 2. How many ATPs can be produced from one molecule of glucoseanaerobically? Aerobically? An aerobically, two ATPs can be produced from one glucose molecule. Aerobically, this figure is 30 to 32, depending on in which tissue it is occurring. 3. What are the different names used to describe the pathway discussed in this chapter? The citric acid cycle is also called the Krebs cycle, the tricarboxylic acid cycle, and the TCA cycle. 4. What is meant by the statement that a pathway is amphibolic? Amphibolic means that the pathway is involved in both catabolism and anabolism. 5. In what part of the cell does the citric acid cycle take place? Does this differ from the part of the cell where glycolysis occurs? The citric acid cycle takes place in the mitochondrial matrix. Glycolysis takes place in the cytosol. 6. How does pyruvate from glycolysis get to the pyruvate dehydrogenase complex? There is a transporter on the inner mitochondrial matrix that allows pyruvate from the cytosol to pass into the mitochondria. 7. What electron acceptors play a role in the citric acid cycle? NAD+ and FAD are the primary electron acceptors of the citric acid cycle.
8. What three molecules produced during the citric acid cycle are an indirect or direct source of high-energy compounds? NADH and FADH2 are indirect sources of energy produced in the TCA cycle. GTP is a direct source of energy. 9. How many enzymes are involved in mammalian pyruvate dehydrogenase? What are their functions? Five enzymes are involved in the pyruvate dehydrogenase complex of mammals. Pyruvate dehydrogenase transfers a two- carbon unit to TPP and releases CO2. Dihydrolipoyl transacetylase transfers the two-carbon acetyl unit to lipoic acid and then to coenzyme A. Dihydrolipoyl dehydrogenase reoxidizes lipoic acid and reduces NAD+ to NADH. Pyruvate dehydrogenase kinase phosphorylates PDH. PDH phosphatase removes the phosphate. 10. Briefly describe the dual role of lipoic acid in the pyruvate dehydrogenase complex. Lipoic acid plays a role both in redox and in acetyl-transfer reactions. 11. What is the advantage to the organization of the PDH complex? Five enzymes are all in close proximity for efficient shuttling of the acetyl unit between molecules and efficient control of the complex by phosphorylation. 12. In the PDH reaction alone, we can see cofactors that come from four different vitamins. What are they? Thiamine pyrophosphate comes from the B vitamin thiamine. Lipoic acid is a vitamin. NAD+ comes from the B vitamin niacin.fad comes from the B vitamin riboflavin.
13. Draw the structures of the activated carbon groups bound to thiamine pyrophosphate in three enzymes that contain this coenzyme. Hint: Keto enol tautomerism may enter into the picture. 14. Prepare a sketch showing how the individual reactions of the three enzymes of the pyruvate dehydrogenase complex give rise to the overall reaction.
15. Why is the reaction catalyzed by citrate synthase considered a condensation reaction? A condensation reaction is one in which a new carbon carbon bond is formed. The reaction of acetyl-coa and oxaloacetate to produce citrate involves formation of such a carbon carbon bond. 16. What does it mean when an enzyme has the name synthase? It means that the reaction catalyzed by the enzyme produces the product that is part of the name and does not require a direct input of energy from a high-energy phosphate. Thus, citrate synthase catalyzes the synthesis of citrate without using ATP to do it.
17. What is fluoroacetate? Why is it used? Fluoroacetate is a poison that is produced naturally in some plants and is also used as a poison against undesirable pests. It is poisonous because it is used by citrate synthase to make fluorocitrate, which is an inhibitor of the citric acid cycle. 18. With respect to stereochemistry, what is unique about the reaction catalyzed by aconitase? The reaction involves an achiral molecule (citrate) being converted to a chiral one (isocitrate). 19. In which steps of the aerobic processing of pyruvate is CO2produced? Conversion of pyruvate to acetyl-coa, conversion of isocitrate to a-ketoglutarate, and conversion of a-ketoglutarate to succinyl-coa. 20. In which steps of the aerobic processing of pyruvate are reduced electron carriers produced? Conversion of pyruvate to acetyl-coa, conversion of isocitrate to a-ketoglutarate, conversion of a-ketoglutarate to succinyl-coa, conversion of succinate to fumarate, and conversion of malate to oxaloacetate. 21. What type of reaction is catalyzed by isocitrate dehydrogenase and a-ketoglutarate dehydrogenase? These enzymes catalyze oxidative decarboxylations. 22. What are the similarities and differences between the reactions catalyzed by pyruvate dehydrogenase and a-ketoglutarate dehydrogenase? The reactions proceed by the same mechanism and use the same cofactors. The difference is the initial substrate, which is pyruvate or a-ketoglutarate. During the course of the
reaction, pyruvate dehydrogenase shuttles an acetyl unit through the reaction while a- ketoglutarate dehydrogenase shuttles a succinyl unit. 23. What does it mean when an enzyme is called a synthetase? A synthetase is an enzyme that synthesizes a molecule and uses a high-energy phosphate in the process. 24. Why can we say that production of a GTP is equivalent to production of an ATP? GTP is equivalent to ATP because an enzyme, nucleoside diphosphate kinase, is able to interconvert GTP and ATP. 25. What are the major differences between the oxidations in the citric acid cycle that use NAD+ as an electron acceptor and the one that uses FAD? The enzymes that reduce NAD+ are all soluble, matrix enzymes, while succinate dehydrogenase is membrane-bound. The NAD+-linked dehydrogenases all catalyze oxidations that involve carbons and oxygens, such as an alcohol group being oxidized to an aldehyde or aldehyde to carboxylic acid. The FAD-linked dehydrogenase oxidizes a carbon carbon single bond to a double bond. 26. ATP is a competitive inhibitor of NADH binding to malate dehydrogenase, as are ADP and AMP. Suggest a structural basis for this inhibition. There is an adenine nucleotide portion in the structure of NADH, with a specific binding site on NADH-linked dehydrogenases for this portion of NADH. 27. Is the conversion of fumarate to malate a redox (electron transfer) reaction? Give the reason for your answer. The conversion of fumarate to malate is a hydration reaction, not a redox reaction.
28. We have seen one of the four possible isomers of isocitrate, the one produced in the aconitase reaction. Draw the configurations of the other three. 29. Show, by Lewis electron-dot structures of the appropriate portions of the molecule, where electrons are lost in the following conversions:(a) Pyruvate to acetyl-coa(b) Isocitrate to a-ketoglutarate(c) a-ketoglutarate to succinyl-coa(d) Succinate to fumarate(e) Malate to oxaloacetate
30. Which steps of aerobic metabolism of pyruvate through the citric acid cycle are control points?
The reactions are catalyzed by pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase. 31. Describe the multiple ways that PDH is controlled. PDH is controlled allosterically. It is inhibited by ATP, acetyl-coa, and NADH. In addition, it is subject to control by phosphorylation. When PDH kinase phosphorylates PDH, it becomes inactive. Removing the phosphate with the PDH phosphatase reactivates it. 32. What are the two most common inhibitors of steps of the citric acid cycle and the reaction catalyzed by pyruvate dehydrogenase? ATP and NADH are the two most common inhibitors. 33. How does an increase in the ADP/ATP ratio affect the activity of isocitrate dehydrogenase? If the amount of ADP in a cell increases relative to the amount of ATP, the cell needs energy (ATP). This situation not only favors the reactions of the citric acid cycle, which release energy, activating isocitrate dehydrogenase, but also stimulates the formation of NADH and FADH2 for ATP production by electron transport and oxidative phosphorylation. 34. How does an increase in the NADH/NAD+ ratio affect the activity of pyruvate dehydrogenase? If the amount of NADH in a cell increases relative to the amount of NAD+, the cell has completed a number of energy-releasing reactions. There is less need for the citric acid cycle to be active; as a result, the activity of pyruvate dehydrogenase is decreased. 35. Would you expect the citric acid cycle to be more or less active when a cell has a high ATP/ADP ratio and a high NADH/NAD+ ratio? Give the reason for your answer.
The citric acid cycle is less active when a cell has a high ATP/ADP ratio and a high NADH/NAD+ ratio. Both ratios indicate a high energy charge in the cell, indicating less of a need for the energy-releasing reactions of the citric acid cycle. 36. Would you expect DG ' for the hydrolysis of a thioester to be (a) large and negative, (b) large and positive,(c) small and negative, or (d) small and positive? Give the reason for your answer. Thioesters are high-energy compounds that play a role in group-transfer reactions; consequently, their DG 9 of hydrolysis is large and negative to provide energy for the reaction. 37. Acetyl-CoA and succinyl-coa are both high-energy thioesters, but their chemical energy is put to different uses. Elaborate. The energy released by hydrolysis of acetyl-coa is needed for the condensation reaction that links the acetyl moiety to oxaloacetate, yielding citrate. The energy released by hydrolysis of succinyl-coa drives the phosphorylation of GDP, yielding GTP. 38. Some reactions of the citric acid cycle are endergonic. Show how the overall cycle is exergonic. (See Table 19.2.) Table 19.2 shows that the sum of the energies of the individual reactions is 244.3 kj (210.6 kcal) for each mole of acetyl-coa that enters the cycle.
39. How could the expression milking it for all it s worth relate to the citric acid cycle? The expression would relate to the intensive extraction of energy from intermediate compounds by redox reactions. Including the pyruvate dehydrogenase reaction, 5 of 9 reactions are redox reactions (in contrast with only 1 of 10 in glycolysis). Accordingly, energy is rapidly extracted from carbon compounds (yielding the energy-less CO2) and is transferred to NAD+ and FAD for subsequent utilization. 40. Using the information in Chapters 17 19, calculate the amount of ATP that can be produced from one molecule of lactose metabolized aerobically through glycolysis and the citric acid cycle. Lactose is a disaccharide of glucose and galactose. There is no energy cost in the hydrolysis of the bond between the two monosaccharides, so essentially there are two hexoses to consider. Because the processing of any of the hexoses yields the same amount of energy, the aerobic processing of lactose would lead to 60 to 64ATPs, depending on the tissue and on the shuttle system used. 41. Which enzymes of the citric acid cycle are missing from the glyoxylate cycle? Isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, and succinyl-coa synthetase. 42. What are the unique reactions of the glyoxylate cycle? The conversion of isocitrate to succinate and glyoxylate catalyzed by isocitrate lyase and the conversion of glyoxylate and acetyl-coa to malate catalyzed by malate synthase. 43. Why is it possible for bacteria to survive on acetic acid as a sole carbon source, but not human beings?
Bacteria that have a glyoxylate cycle can convert the acetic acid to amino acids, carbohydrates, and lipids, but humans can use the acetic acid only as an energy source or to make lipids. 44. Describe the various purposes of the citric acid cycle. The citric acid cycle is the central metabolic pathway and indirect producer of energy. It receives fuels from the other pathways at many points and generates reduced electron carriers that go into the electron transport chain. It is also involved in anabolism, as many of its intermediates can be drawn off to synthesize other compounds. 45. The intermediates of glycolysis are phosphorylated, but those of the citric acid cycle are not. Suggest a reason why. The citric acid cycle occurs in the mitochondrial matrix, which is more selective in its permeability than the plasma membrane. 46. Discuss oxidative decarboxylation, using a reaction from this chapter to illustrate your points. In oxidative decarboxylation, the molecule that is oxidized loses a carboxyl group as carbon dioxide. Examples of oxidative decarboxylation include the conversion of pyruvate to acetyl-coa, isocitrate to a-ketoglutarate, and a-ketoglutarate to succinyl- CoA. 47. Many soft drinks contain citric acid as a significant part of their flavor. Is this a good nutrient? Yes, not only is citric acid completely degraded to carbon dioxide and water, but it is also readily absorbed into the mitochondrion.
48. NADH is an important coenzyme in catabolic processes, whereas NADPH appears in anabolic processes. Explain how an exchange of the two can be effected. The following series of reactions exchanges NADH for NADPH. 49. What are the anaplerotic reactions in mammals? A variety of reactions in which amino acids are converted to citric acid cycle intermediates are considered anaplerotic. In addition, pyruvate 1 CO2 can form oxaloacetate via pyruvate carboxylase. 50. Why is acetyl-coa considered the central molecule of metabolism? Many compounds can form acetyl-coa, such as fats, carbohydrates, and many amino acids. Acetyl-CoA can also form fats and ketone bodies, as well as feed directly into the citric acid cycle. 51. Why is the citric acid cycle considered part of aerobic metabolism, even though molecular oxygen does not appear in any reaction? The NADH and FADH2 produced by the citric acid cycle are the electron donors in the electron transport chain linked to oxygen. Because of this connection, the citric acid cycle is considered part of aerobic metabolism.