19.7 What Are the Anaplerotic, or “Filling Up,”Reactions? 583 H + + C H 2 C H 2 O + P i O COO – COO – C H 2 C O COO – COO – C H 2 C COO – COO – H OH C CH 3 O COO – C CH 2 2 – O 3 PO COO – NADPH NADP + P i C CH 3 O COO – ATP ADP + CO 2 CO 2 CO 2 Phosphoenolpyruvate (PEP) PEP carboxylase Oxaloacetate Oxaloacetate Pyruvate Pyruvate Pyruvate carboxylase Malic enzyme L-Malate FIGURE 19.17 Pyruvate carboxylase, phosphoenol- pyruvate (PEP) carboxylase, and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle inter- mediates. A DEEPER LOOK Fool’s Gold and the Reductive Citric Acid Cycle—The First Metabolic Pathway? How did life arise on the planet Earth? It was once supposed that a reducing atmosphere, together with random synthesis of organic compounds, gave rise to a prebiotic “soup,” in which the first living things appeared. However, certain key compounds, such as arginine, lysine, and histidine; the straight-chain fatty acids; porphyrins; and essential coenzymes, have not been convincingly synthesized under simulated prebiotic conditions. This and other problems have led re- searchers to consider other models for the evolution of life. One of these alternative models, postulated by Günter Wächtershäuser, involves an archaic version of the TCA cycle running in the reverse (reductive) direction. Reversal of the TCA cycle results in assimilation of CO 2 and fixation of carbon as shown. For each turn of the reversed cycle, two carbons are fixed in the formation of isocitrate and two more are fixed in the re- ductive transformation of acetyl-CoA to oxaloacetate. Thus, for every succinate that enters the reversed cycle, two succinates are returned, making the cycle highly autocatalytic. Because TCA cycle intermediates are involved in many biosynthetic pathways (see Section 19.6), a reversed TCA cycle would be a bountiful and broad source of metabolic substrates. A reversed, reductive TCA cycle would require energy input to drive it. What might have been the thermodynamic driving force for such a cycle? Wächtershäuser hypothesizes that the anaerobic reaction of FeS and H 2 S to form insoluble FeS 2 (pyrite, also known as fool’s gold) in the prebiotic milieu could have been the driving reaction: FeS ϩ H 2 S⎯⎯→FeS 2 (pyrite) ↓ ϩ H 2 This reaction is highly exergonic, with a standard-state free energy change (⌬G°Ј) of Ϫ38 kJ/mol. Under the conditions that might have existed in a prebiotic world, this reaction would have been sufficiently exergonic to drive the reductive steps of a reversed TCA cycle. Wächtershäuser has also suggested that early metabolic processes first occurred on the surface of pyrite and other related mineral ma- terials. The iron–sulfur chemistry that prevailed on these mineral surfaces may have influenced the evolution of the iron–sulfur pro- teins that control and catalyze many reactions in modern pathways (including the succinate dehydrogenase and aconitase reactions of the TCA cycle). This reductive citric acid cycle has been shown to oc- cur in certain extant archaea and bacteria, where it serves all their carbon needs. Oxaloacetate Oxaloacetate Citrate Isocitrate ␣-Ketoglutarate Succinyl CoA Succinate Succinate Fumarate Fumarate Malate Pyruvate Malate XH 2 X XH 2 X XH 2 X XH 2 X XH 2 X XH 2 X PEP Acetyl-CoA CO 2 CO 2 CO 2 CO 2 ᮡ A reductive, reversed TCA cycle. 584 Chapter 19 The Tricarboxylic Acid Cycle It is worth noting that the reaction catalyzed by PEP carboxykinase could also function as an anaplerotic reaction, were it not for the particular properties of the enzyme. CO 2 binds weakly to PEP carboxykinase, whereas oxaloacetate binds very tightly (K D ϭ 2 ϫ 10 Ϫ6 M), and, as a result, the enzyme favors formation of PEP from oxaloacetate. The catabolism of amino acids provides pyruvate, acetyl-CoA, oxaloacetate, fumarate, ␣-ketoglutarate, and succinate, all of which may be oxidized by the TCA cycle. In this way, proteins may serve as excellent sources of nutrient energy, as seen in Chapter 25. 19.8 How Is the TCA Cycle Regulated? Situated as it is between glycolysis and the electron-transport chain, the TCA cycle must be carefully controlled. If the cycle were permitted to run unchecked, large amounts of metabolic energy could be wasted in overproduction of reduced coenzymes and ATP; conversely, if it ran too slowly, ATP would not be produced rapidly enough to sat- isfy the needs of the cell. Also, as just seen, the TCA cycle is an important source of pre- cursors for biosynthetic processes and must be able to provide them as needed. What are the sites of regulation in the TCA cycle? Based on our experience with glycolysis (see Figure 18.22), we might anticipate that some of the reactions of the TCA cycle would operate near equilibrium under cellular conditions (with ⌬G Ͻ 0), whereas others—the sites of regulation—would be characterized by large negative ⌬G values. Estimates for the values of ⌬G in mitochondria, based on mitochondrial concentrations of metabolites, are summarized in Table 19.1. Three reactions of the cycle—citrate synthase, isocitrate dehydrogenase, and ␣-ketoglutarate dehydrogenase—operate with large negative ⌬G values under mitochondrial conditions and are thus the primary sites of regulation in the cycle. The regulatory actions that control the TCA cycle are shown in Figure 19.18. As one might expect, the principal regulatory “signals” are the concentrations of acetyl- CoA, ATP, NAD ϩ , and NADH, with additional effects provided by several other metabolites. The main sites of regulation are pyruvate dehydrogenase, citrate syn- thase, isocitrate dehydrogenase, and ␣-ketoglutarate dehydrogenase. All of these en- zymes are inhibited by NADH, so when the cell has produced all the NADH that can conveniently be turned into ATP, the cycle shuts down. For similar reasons, ATP is an inhibitor of pyruvate dehydrogenase and isocitrate dehydrogenase. The TCA cycle is turned on, however, when either the ADP/ATP or NAD ϩ /NADH ratio is high, an indication that the cell has run low on ATP or NADH. Regulation of the TCA cycle by NADH, NAD ϩ , ATP, and ADP thus reflects the energy status of the cell. On the other hand, succinyl-CoA is an intracycle regulator, inhibiting citrate syn- thase and ␣-ketoglutarate dehydrogenase. Acetyl-CoA acts as a signal to the TCA cycle that glycolysis or fatty acid breakdown is producing two-carbon units. Acetyl- CoA activates pyruvate carboxylase, the anaplerotic reaction that provides oxaloac- etate, the acceptor for increased flux of acetyl-CoA into the TCA cycle. Pyruvate Dehydrogenase Is Regulated by Phosphorylation/ Dephosphorylation As we shall see in Chapter 22, most organisms can synthesize sugars such as glucose from pyruvate. However, animals cannot synthesize glucose from acetyl-CoA. For this reason, the pyruvate dehydrogenase complex, which converts pyruvate to + C CH 2 2 – O 3 PO COO – C H 2 C O COO – GDP GTP COO – CO 2 PEP Oxaloacetate 19.8 How Is the TCA Cycle Regulated? 585 acetyl-CoA, plays a pivotal role in metabolism. Conversion to acetyl-CoA commits nutrient carbon atoms either to oxidation in the TCA cycle or to fatty acid synthe- sis (see Chapter 24). Because this choice is so crucial to the organism, pyruvate de- hydrogenase is a carefully regulated enzyme. It is subject to product inhibition and is further regulated by nucleotides. Finally, activity of pyruvate dehydrogenase is regulated by phosphorylation and dephosphorylation of the enzyme complex itself. High levels of either product, acetyl-CoA or NADH, allosterically inhibit the pyruvate dehydrogenase complex. Acetyl-CoA specifically blocks dihydrolipoyl transacetylase, and NADH acts on dihydrolipoyl dehydrogenase. The mammalian pyruvate dehydrogenase is also regulated by covalent modifications. As shown in Figure 19.19, a Mg 2ϩ -dependent pyruvate dehydrogenase kinase is associated with the enzyme in mammals. This kinase is allosterically activated by NADH and acetyl- CoA, and when levels of these metabolites rise in the mitochondrion, they stimulate phosphorylation of a serine residue on the pyruvate dehydrogenase subunit, block- ing the first step of the pyruvate dehydrogenase reaction, the decarboxylation of pyruvate. Inhibition of the dehydrogenase in this manner eventually lowers the lev- els of NADH and acetyl-CoA in the matrix of the mitochondrion. Reactivation of the enzyme is carried out by pyruvate dehydrogenase phosphatase, a Ca 2ϩ -activated enzyme that binds to the dehydrogenase complex and hydrolyzes the phosphoser- ine moiety on the dehydrogenase subunit. At low ratios of NADH to NAD ϩ and low Pyruvate carboxylase Citrate synthase Isocitrate dehydrogenase ␣-Ketoglutarate dehydrogenase Pyruvate dehydrogenase + + + + + Acetyl-CoA Pyruvate Oxaloacetate Succinyl-CoA Citrate Isocitrate Acetyl-CoA Acetyl-CoA , Succinyl-CoA TCA Cycle ␣-Ketoglutarate Succinate Fumarate Malate ADP ATP NAD + ATP ATP ATP Succinyl-CoA AMP NADH NADH P i ADP NADH NADH CoA H 2 O H 2 O CO 2 CO 2 CO 2 CO 2 GDP P i GTP FIGURE 19.18 Regulation of the TCA cycle. 586 Chapter 19 The Tricarboxylic Acid Cycle acetyl-CoA levels, the phosphatase maintains the dehydrogenase in an activated state, but a high level of acetyl-CoA or NADH once again activates the kinase and leads to the inhibition of the dehydrogenase. Insulin and Ca 2ϩ ions activate de- phosphorylation, and pyruvate inhibits the phosphorylation reaction. Pyruvate dehydrogenase is also sensitive to the energy status of the cell. AMP ac- tivates pyruvate dehydrogenase, whereas GTP inhibits it. High levels of AMP are a sign that the cell may become energy-poor. Activation of pyruvate dehydrogenase under such conditions commits pyruvate to energy production. Isocitrate Dehydrogenase Is Strongly Regulated The mechanism of regulation of isocitrate dehydrogenase is in some respects the reverse of pyruvate dehydrogenase. The mammalian isocitrate dehydrogenase is subject only to allosteric activation by ADP and NAD ϩ and to inhibition by ATP and NADH. Thus, high NAD ϩ /NADH and ADP/ATP ratios stimulate isocitrate dehy- drogenase and TCA cycle activity. It may seem surprising that isocitrate dehydrogenase is strongly regulated, be- cause it is not an apparent branch point within the TCA cycle. However, the citrate/ isocitrate ratio controls the rate of production of cytosolic acetyl-CoA, because acetyl- CoA in the cytosol is derived from citrate exported from the mitochondrion. (Break- down of cytosolic citrate produces oxaloacetate and acetyl-CoA, which can be used in a variety of biosynthetic processes.) Thus, isocitrate dehydrogenase activity in the mitochondrion favors catabolic TCA cycle activity over anabolic utilization of acetyl- CoA in the cytosol. Interestingly, the Escherichia coli isocitrate dehydrogenase is regulated by cova- lent modification. Serine residues on each subunit of the dimeric enzyme are phosphorylated by a protein kinase, causing inhibition of the isocitrate dehydro- genase activity. Activity is restored by the action of a specific phosphatase. When TCA cycle and glycolytic intermediates—such as isocitrate, 3-phosphoglycerate, pyruvate, PEP, and oxaloacetate—are high, the kinase is inhibited, the phos- phatase is activated, and the TCA cycle operates normally. When levels of these intermediates fall, the kinase is activated, isocitrate dehydrogenase is inhibited, and isocitrate is diverted to the glyoxylate pathway, as explained in the next section. High NADH/NAD + ratio High AcCoA/CoASH ratio Low NADH/NAD + ratio Low AcCoA/CoASH ratio ATP ADP Pyruvate dehydrogenase kinase P i H 2 O Ca 2+ Pyruvate dehydrogenase phosphatase Active pyruvate dehydrogenase Inactive pyruvate dehydrogenase FIGURE 19.19 Regulation of the pyruvate dehydroge- nase reaction. 19.9 Can Any Organisms Use Acetate as Their Sole Carbon Source? 587 19.9 Can Any Organisms Use Acetate as Their Sole Carbon Source? Plants (particularly seedlings, which cannot yet accomplish efficient photosynthe- sis), as well as some bacteria and algae, can use acetate as the only source of carbon for all the carbon compounds they produce. Although we saw that the TCA cycle can supply intermediates for some biosynthetic processes, the cycle gives off 2 CO 2 for every two-carbon acetate group that enters and cannot effect the net synthesis of TCA cycle intermediates. Thus, it would not be possible for the cycle to produce the massive amounts of biosynthetic intermediates needed for acetate-based growth unless alternative reactions were available. In essence, the TCA cycle is geared pri- marily to energy production, and it “wastes” carbon units by giving off CO 2 . Modi- fication of the cycle to support acetate-based growth would require eliminating the CO 2 -producing reactions and enhancing the net production of four-carbon units (that is, oxaloacetate). Plants and bacteria employ a modification of the TCA cycle called the glyoxylate cycle to produce four-carbon dicarboxylic acids (and eventu- ally even sugars) from two-carbon acetate units. The glyoxylate cycle bypasses the two oxidative decarboxylations of the TCA cycle and instead routes isocitrate through the isocitrate lyase and malate synthase reactions (Figure 19.20). Glyoxy- late produced by isocitrate lyase reacts with a second molecule of acetyl-CoA to form L-malate. The net effect is to conserve carbon units, using two acetyl-CoA molecules per cycle to generate oxaloacetate. Some of this is converted to PEP and then to glu- cose by pathways discussed in Chapter 22. C SCoA O H 3 C H 2 C C O COO – COO – COO – H 2 C COO – H C HO C SCoA O H 3 C O HC COO – H 2 C COO – H 2 C COO – H 2 C COO – HC COO – HC OH COO – H 2 C COO – C COO – H 2 C COO – HO Acetyl-CoA GLYOXYLATE CYCLE Isocitrate lyase H 2 O CoASH Oxaloacetate Malate Malate synthase CoASH Acetyl-CoA Glyoxylate Succinate Isocitrate Citrate FIGURE 19.20 The glyoxylate cycle.The first two steps are identical to TCA cycle reactions.The third step bypasses the CO 2 -evolving steps of the TCA cycle to produce succi- nate and glyoxylate.The malate synthase reaction forms malate from glyoxylate and another acetyl-CoA.The re- sult is that one turn of the cycle consumes one oxalo- acetate and two acetyl-CoA molecules but produces two molecules of oxaloacetate. (Succinate produced in the isocitrate lyase reaction is converted to oxaloacetate by TCA cycle reactions.) The net for this cycle is one oxalo- acetate from two acetyl-CoA molecules. 588 Chapter 19 The Tricarboxylic Acid Cycle The Glyoxylate Cycle Operates in Specialized Organelles The enzymes of the glyoxylate cycle in plants are contained in glyoxysomes, or- ganelles devoted to this cycle. Yeast and algae carry out the glyoxylate cycle in the cytoplasm. The enzymes common to both the TCA and glyoxylate pathways exist as isozymes, with spatially and functionally distinct enzymes operating independently in the two cycles. Isocitrate Lyase Short-Circuits the TCA Cycle by Producing Glyoxylate and Succinate The isocitrate lyase reaction (Figure 19.21) produces succinate, a four-carbon product of the cycle, as well as glyoxylate, which can then combine with a second molecule of acetyl-CoA. Isocitrate lyase catalyzes an aldol cleavage and is similar to the reaction mediated by aldolase in glycolysis. The malate synthase reaction (see Figure 19.20), a Claisen condensation of acetyl-CoA with the aldehyde of glyoxylate to yield malate, is quite similar to the citrate synthase reaction. Compared with the TCA cycle, the glyoxylate cycle (1) contains only five steps (as opposed to eight), (2) lacks the CO 2 -liberating reactions, (3) consumes two molecules of acetyl-CoA per cycle, and (4) produces four-carbon units (oxaloacetate) as opposed to one- carbon units. The Glyoxylate Cycle Helps Plants Grow in the Dark The existence of the glyoxylate cycle explains how certain seeds grow underground (or in the dark), where photosynthesis is impossible. Many seeds (peanuts, soy- beans, and castor beans, for example) are rich in lipids, and as we will see in Chap- ter 23, most organisms degrade the fatty acids of lipids to acetyl-CoA. Glyoxysomes form in seeds as germination begins, and the glyoxylate cycle uses the acetyl-CoA produced in fatty acid oxidation to provide large amounts of oxaloacetate and other intermediates for carbohydrate synthesis. Once the growing plant begins photosynthesis and can fix CO 2 to produce carbohydrates (see Chapter 21), the glyoxysomes disappear. Glyoxysomes Must Borrow Three Reactions from Mitochondria Glyoxysomes do not contain all the enzymes needed to run the glyoxylate cycle: Succinate dehydrogenase, fumarase, and malate dehydrogenase are absent. Conse- quently, glyoxysomes must cooperate with mitochondria to run their cycle (Figure 19.22). Succinate travels from the glyoxysomes to the mitochondria, where it is con- verted to oxaloacetate. Transamination to aspartate follows because oxaloacetate can- not be transported out of the mitochondria. Aspartate formed in this way then moves from the mitochondria back to the glyoxysomes, where a reverse transamination with ␣-ketoglutarate forms oxaloacetate, completing the shuttle. Finally, to balance the transaminations, glutamate shuttles from glyoxysomes to mitochondria. + E C COO – B B + H HC O COO – H 2 C B H + COO – H 2 C COO – E B H 2 C H COO – CH COO – O H 2R, 3S-Isocitrate Glyoxylate Succinate FIGURE 19.21 The isocitrate lyase reaction. Summary 589 Glyoxysome Mitochondrion Aspartate ␣-Keto- glutarate Glutamate Oxaloacetate ␣-Keto- glutarate Aspartate Glutamate Oxaloacetate Acetyl- CoA Acetyl- CoA Malate Malate Oxaloacetate Phosphoenolpyruvate Carbohydrate Cytosol Malate Fumarate TCA Succinate Succinate Citrate Isocitrate Fatty acids Glyoxylate Glyoxylate cycle FIGURE 19.22 Glyoxysomes lack three of the enzymes needed to run the glyoxylate cycle. Succinate dehy- drogenase, fumarase, and malate dehydrogenase are all “borrowed”from the mitochondria in a shuttle in which succinate and glutamate are passed to the mitochondria and ␣-ketoglutarate and aspartate are passed to the glyoxysome. SUMMARY The glycolytic pathway converts glucose to pyruvate and produces two molecules of ATP per glucose—only a small fraction of the potential energy available from g lucose. In the presence of oxygen, pyruvate is oxidized to CO 2 , releasing the rest of the energy available from glucose via the TCA cycle. 19.1 What Is the Chemical Logic of the TCA Cycle? The entry of new carbon units into the cycle is through acetyl-CoA. Transfer of the two- carbon acetyl group from acetyl-CoA to the four-carbon oxaloacetate to yield six-carbon citrate is catalyzed by citrate synthase. A dehydra- tion–rehydration rearrangement of citrate yields isocitrate. Two suc- cessive decarboxylations produce ␣-ketoglutarate and then succinyl- CoA, a CoA conjugate of a four-carbon unit. Several steps later, oxaloacetate is regenerated and can combine with another two-carbon unit of acetyl-CoA. 19.2 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA? The pyruvate dehydrogenase complex (PDC) is a noncovalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA. 19.3 How Are Two CO 2 Molecules Produced from Acetyl-CoA? Citrate synthase combines acetyl-CoA with oxaloacetate in a Perkin condensa- tion (a carbon–carbon condensation between a ketone or aldehyde and an ester). A general base on the enzyme accepts a proton from the methyl group of acetyl-CoA, producing a stabilized ␣-carbanion of acetyl- CoA. This strong nucleophile attacks the ␣-carbonyl of oxaloacetate, yielding citryl-CoA, which is hydrolyzed to citrate and CoASH. Citrate is isomerized to isocitrate by aconitase in a two-step process involving aconitate as an intermediate. The elements of water are first abstracted from citrate to yield aconitate, which is then rehydrated with HO and HOO adding back in opposite positions to produce isocitrate. The net effect is the conversion of a tertiary alcohol (citrate) to a sec- ondary alcohol (isocitrate). The two-step isocitrate dehydrogenase reaction involves (1) oxida- tion of the C-2 alcohol of isocitrate to form oxalosuccinate, followed by (2) a -decarboxylation reaction that expels the central carboxyl group as CO 2 , leaving the product ␣-ketoglutarate. Oxalosuccinate, the -keto acid produced by the initial dehydrogenation reaction, is unstable and thus is readily decarboxylated. ␣-Ketoglutarate dehydrogenase is a multienzyme complex—consisting of ␣-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase—that employs five different coenzymes. The dihydrolipoyl dehydrogenase in this reaction is identical to that in the pyruvate dehydrogenase reaction. The mechanism is an oxidative decar- boxylation analogous to that of pyruvate dehydrogenase. Succinyl-CoA is the product. 19.4 How Is Oxaloacetate Regenerated to Complete the Cycle? Succinyl-CoA synthetase catalyzes a substrate-level phosphorylation: Succinyl-CoA is a high-energy intermediate and is used to drive the phosphorylation of GDP to GTP (in mammals) or ADP to ATP (in plants and bacteria). Succinate dehydrogenase (succinate–coenzyme Q reductase of the electron-transport chain) catalyzes removal of H atoms across a COC bond and produces the trans-unsaturated fumarate. Fumarate is hydrated in a stereospecific reaction by fumarase to give L-malate. The reaction involves trans-addition of the elements of water across the double bond. 590 Chapter 19 The Tricarboxylic Acid Cycle Malate dehydrogenase completes the TCA cycle. This reaction is very endergonic, with a ⌬G°Ј of ϩ30 kJ/mol. Consequently, the concentra- tion of oxaloacetate in the mitochondrial matrix is usually quite low. Ox- idation of malate to oxaloacetate is coupled to reduction of yet another molecule of NAD ϩ , the third one of the cycle. 19.5 What Are the Energetic Consequences of the TCA Cycle? The cycle is exergonic, with a net ⌬G°Ј for one pass around the cycle of approximately Ϫ40 kJ/mol. Three NADH, one [FADH 2 ], and one ATP equivalent are produced in each turn of the cycle. 19.6 Can the TCA Cycle Provide Intermediates for Biosynthesis? ␣-Ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate are all pre- cursors of important cellular species. A transamination reaction con- verts ␣-ketoglutarate directly to glutamate, which can then serve as a precursor for proline, arginine, and glutamine. Succinyl-CoA provides most of the carbon atoms of the porphyrins. Oxaloacetate can be transaminated to produce aspartate. Aspartic acid itself is a precursor of the pyrimidine nucleotides and, in addition, is a key precursor for the synthesis of asparagine, methionine, lysine, threonine, and isoleucine. Oxaloacetate can also be decarboxylated to yield PEP, which is a key element of several pathways. 19.7 What Are the Anaplerotic, or “Filling Up,” Reactions? Ana- plerotic reactions replenish the TCA cycle intermediates. Examples in- clude PEP carboxylase and pyruvate carboxylase, both of which synthe- size oxaloacetate from pyruvate. 19.8 How Is the TCA Cycle Regulated? The main sites of regulation are pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and ␣-ketoglutarate dehydrogenase. All of these enzymes are inhibited by NADH. ATP is an inhibitor of pyruvate dehydrogenase and isocitrate dehydrogenase. The TCA cycle is turned on, however, when either the ADP/ATP or NAD ϩ /NADH ratio is high. Regulation of the TCA cycle by NADH, NAD ϩ , ATP, and ADP thus reflects the energy status of the cell. Succinyl-CoA is an intracycle regulator, inhibiting citrate synthase and ␣-ketoglutarate dehydrogenase. Acetyl-CoA activates pyruvate car- boxylase, the anaplerotic reaction that provides oxaloacetate, the ac- ceptor for acetyl-CoA entry into the TCA cycle. 19.9 Can Any Organisms Use Acetate as Their Sole Carbon Source? Plants and bacteria employ a modification of the TCA cycle called the glyoxylate cycle to produce four-carbon dicarboxylic acids (and eventu- ally even sugars) from two-carbon acetate units. The glyoxylate cycle by- passes the two oxidative decarboxylations of the TCA cycle and instead routes isocitrate through the isocitrate lyase and malate synthase reac- tions. Glyoxylate produced by isocitrate lyase reacts with a second mol- ecule of acetyl-CoA to form L-malate. The net effect is to conserve carbon units, using two acetyl-CoA molecules per cycle to generate oxaloacetate. PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. Describe the labeling pattern that would result from the introduc- tion into the TCA cycle of glutamate labeled at C ␥ with 14 C. 2. Describe the effect on the TCA cycle of (a) increasing the con- centration of NAD ϩ , (b) reducing the concentration of ATP, and (c) increasing the concentration of isocitrate. 3. (Integrates with Chapter 15.) The serine residue of isocitrate dehy- drogenase that is phosphorylated by protein kinase lies within the active site of the enzyme. This situation contrasts with most other examples of covalent modification by protein phosphorylation, where the phosphorylation occurs at a site remote from the active site. What direct effect do you think such active-site phosphoryla- tion might have on the catalytic activity of isocitrate dehydrogenase? (See Barford, D., 1991. Molecular mechanisms for the control of enzymic activity by protein phosphorylation. Biochimica et Biophysica Acta 1133:55–62.) 4. The first step of the ␣-ketoglutarate dehydrogenase reaction involves decarboxylation of the substrate and leaves a covalent TPP interme- diate. Write a reasonable mechanism for this reaction. 5. In a tissue where the TCA cycle has been inhibited by fluoroacetate, what difference in the concentration of each TCA cycle metabolite would you expect, compared with a normal, uninhibited tissue? 6. On the basis of the descriptions of the physical properties of FAD and FADH 2 , suggest a method for the measurement of the enzyme activity of succinate dehydrogenase. 7. Starting with citrate, isocitrate, ␣-ketoglutarate, and succinate, state which of the individual carbons of the molecule undergo oxidation in the next step of the TCA cycle. Which molecules undergo a net oxidation? 8. In addition to fluoroacetate, consider whether other analogs of TCA cycle metabolites or intermediates might be introduced to in- hibit other, specific reactions of the cycle. Explain your reasoning. 9. Based on the action of thiamine pyrophosphate in catalysis of the pyruvate dehydrogenase reaction, suggest a suitable chemical mechanism for the pyruvate decarboxylase reaction in yeast: Pyruvate ⎯⎯→ acetaldehyde ϩ CO 2 10. (Integrates with Chapter 3.) Aconitase catalyzes the citric acid cycle reaction: Citrate 34 isocitrate The standard free energy change, ⌬G°Ј, for this reaction is ϩ6.7 kJ/mol. However, the observed free energy change (⌬G) for this re- action in pig heart mitochondria is ϩ0.8 kJ/mol. What is the ratio of [isocitrate]/[citrate] in these mitochondria? If [isocitrate] ϭ 0.03 mM, what is [citrate]? 11. Describe the labeling pattern that would result if 14 CO 2 were incor- porated into the TCA cycle via the pyruvate carboxylase reaction. 12. Describe the labeling pattern that would result if the reductive, reversed TCA cycle (see A Deeper Look on page 583) operated with 14 CO 2 . 13. Describe the labeling pattern that would result in the glyoxylate cycle if a plant were fed acetyl-CoA labeled at the OCH 3 carbon. 14. The malate synthase reaction, which produces malate from acetyl- CoA and glyoxylate in the glyoxylate pathway, involves chemistry sim- ilar to the citrate synthase reaction. Write a mechanism for the malate synthase reaction and explain the role of CoA in this reaction. 15. In most cells, fatty acids are synthesized from acetate units in the cy- tosol. However, the primary source of acetate units is the TCA cycle in mitochondria, and acetate cannot be transported directly from the mitochondria to the cytosol. Cells solve this problem by exporting cit- rate from the mitochondria and then converting citrate to acetate and oxaloacetate. Then, because cells cannot transport oxaloacetate into mitochondria directly, they must convert it to malate or pyruvate, both of which can be taken up by mitochondria. Draw a complete pathway for citrate export, conversion of citrate to malate and pyru- vate, and import of malate and pyruvate by mitochondria. a. Which of the reactions in this cycle might require energy input? b. What would be the most likely source of this energy? c. Do you recognize any of the enzyme reactions in this cycle? d. What coenzymes might be required to run this cycle? 16. A typical intramitochondrial concentration of malate is 0.22 mM. If the ratio of NAD ϩ to NADH in mitochondria is 20, and if the malate dehydrogenase reaction is at equilibrium, calculate the concentra- tion of oxaloacetate in the mitochondrion at 20°C. A typical mito- Further Reading 591 chondrion can be thought of as a cylinder 1 m in diameter and 2 m in length. Calculate the number of molecules of oxaloacetate in a mitochondrion. In analogy with pH (the negative logarithm of [H ϩ ]), what is pOAA? 17. Glycolysis, the pyruvate dehydrogenase reaction, and the TCA cycle result in complete oxidation of a molecule of glucose to CO 2 . Re- view the calculation of oxidation numbers for individual atoms in any molecule, and then calculate the oxidation numbers of the car- bons of glucose, pyruvate, the acetyl carbons of acetyl-CoA, and the metabolites of the TCA cycle to convince yourself that complete ox- idation of glucose involves removal of 24 electrons and that each acetyl-CoA through the TCA cycle gives up 8 electrons. 18. Recalling all reactions of the TCA cycle can be a challenging propo- sition. One way to remember these is to begin with the simplest mol- ecule—succinate, which is a symmetric four-carbon molecule. Be- gin with succinate, and draw the eight reactions of the TCA cycle. Remember that succinate ⎯⎯→ oxaloacetate is accomplished by a special trio of reactions: oxidation of a single bond to a double bond, hydration across the double bond, and oxidation of an alco- hol to a ketone. From there, a molecule of acetyl-CoA is added. If you remember the special function of acetyl-CoA (see A Deeper Look, page 570), this is an easy reaction to draw. From there, you need only isomerize, carry out the two oxidative decarboxylations, and remove the CoA molecule to return to succinate. 19. Aconitase is rapidly inactivated by 2R, 3R-fluorocitrate, which is pro- duced from fluoroacetate in the citrate synthase reaction. Interest- ingly, inactivation by fluorocitrate is accompanied by stoichiometric release of fluoride ion (i.e., one F-ion is lost per aconitase active site). This observation is consistent with “mechanism-based inacti- vation” of aconitase by fluorocitrate. Suggest a mechanism for this inactivation, based on formation of 4-hydroxy-trans-aconitate, which remains tightly bound at the active site. To assess your answer, con- sult this reference: Lauble, H., Kennedy, M., et al., 1996. The reac- tion of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex. Proceedings of the National Academy of Sci- ences 93:13699–13703. Preparing for the MCAT Exam 20. Complete oxidation of a 16-carbon fatty acid can yield 129 mol- ecules of ATP. Study Figure 19.2 and determine how many ATP molecules would be generated if a 16-carbon fatty acid were metabolized solely by the TCA cycle, in the form of 8 acetyl-CoA molecules. 21. Study Figure 19.18 and decide which of the following statements is false? a. Pyruvate dehydrogenase is inhibited by NADH. b. Pyruvate dehydrogenase is inhibited by ATP. c. Citrate synthase is inhibited by NADH. d. Succinyl-CoA activates citrate synthase. e. Acetyl-CoA activates pyruvate carboxylase. FURTHER READING General Bodner, G. M., 1986. The tricarboxylic acid (TCA), citric acid or Krebs cycle. Journal of Chemical Education 63:673–677. Dalsgaard, M. K., 2006. Fuelling cerebral activity in exercising man. Journal of Cerebral Blood Flow and Metabolism 26:731–750. Fisher, C. R., and Girguis, P., 2007. A proteomic snapshot of life at a vent. Science 315:198–199. Gibala, M. J., Young, M. E., et al., 2000. Anaplerosis of the citric acid cycle: Role in energy metabolism of heart and skeletal muscle. Acta Physiologica Scandinavica 168:657–665. Holmes, F. L., 1993. Hans Krebs: Architect of Intermediary Metabolism, 1933- 1937, Vol. 2. Oxford: Oxford University Press. Hu, Y., and Holden, J. F., 2006. Citric acid cycle in the hyperthermo- philic archaeon Pyrobaculum islandicum grown autotrophically, het- erotrophically, and mixotrophically with acetate. Journal of Bacteriol- ogy 188:4350–4355. Krebs, H. A., 1981. Reminiscences and Reflections. Oxford: Oxford Univer- sity Press. Newsholme, E. A., and Leech, A. R., 1983. Biochemistry for the Medical Sci- ences. New York: John Wiley and Sons. Schurr, A., 2006. Lactate: The ultimate cerebral oxidative energy sub- strate? Journal of Cerebral Blood Flow and Metabolism 26:142–152. Smith, E., and Morowitz, H. J., 2004. Universality in intermediary me- tabolism. Proceedings of the National Academy of Sciences U.S.A. 101: 13168–13173. Enzymes of the TCA Cycle Fraser, M. E., James, M. N. G., et al., 1999. A detailed structural descrip- tion of Escherichia coli succinyl-CoA synthetase. Journal of Molecular Biology 285:1633–1653. Perham, R. N., 2000. Swinging arms and swinging domains in multi- functional enzymes: Catalytic machines for multistep reactions. An- nual Review of Biochemistry 69:961–1004. St. Maurice, M., Reinhardt, L., et al., 2007. Domain architecture of pyru- vate carboxylase, a biotin-dependent multifunctional enzyme. Sci- ence 317:1076–1079. Diseases of the TCA Cycle Briere, J J., Favier, J., et al., 2006. Tricarboxylic acid cycle dysfunction as a cause of human diseases and tumor formation. American Journal of Physiology and Cellular Physiology 291:C1114–C1120. Pithukpakorn, M., 2005. Disorders of pyruvate metabolism and the tri- carboxylic acid cycle. Molecular Genetics and Metabolism 85:243–246. Regulation of the TCA Cycle Atkinson, D. E., 1977. Cellular Energy Metabolism and Its Regulation. New York: Academic Press. Bott, M., 2007. Offering surprises: TCA cycle regulation in Corynebac- terium glutamicum. Trends in Microbiology 15:417–425. Gibson, D., and Harris, R., 2001. Metabolic Regulation in Mammals. New York: Taylor and Francis. Pyruvate Dehydrogenase Brautigam, C. A., Wynn, R. M., et al., 2006. Structural insight into in- teractions between dihydrolipoamide dehydrogenase (E3) and E3- binding protein of human pyruvate dehydrogenase complex. Str uc- ture 14:611–621. Harris, R. A., Bowker-Kinley, M. M., et al., 2002. Regulation of the activ- ity of the pyruvate dehydrogenase complex. Advances in Enzyme Reg- ulation 42:249–259. Milne, J. L. S., Shi, D., et al., 2002. Molecular architecture and mecha- nism of an icosahedral pyruvate dehydrogenase complex: A multi- functional catalytic machine. EMBO Journal 21:5587–5598. Sugden, M. C., and Holdness, M. J., 2006. Mechanisms underlying reg- ulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases. Archives of Physiology and Biochemistry 112: 139–149. Zhou, Z. H., McCarthy, D. B., et al., 2001. The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes. Proceedings of the National Academy of Sciences U.S.A. 98: 14802–14807. Glyoxylate Cycle Eastmond, P. J., and Graham, I. A., 2001. Re-examining the role of the glyoxylate cycle in oilseeds. Trends in Plant Science 6:72–77. George Rhoads/Rock Stream Studios 20 Electron Transport and Oxidative Phosphorylation Whereas ATP made in glycolysis and the TCA cycle is the result of substrate- level phosphorylation, NADH-dependent ATP synthesis is the result of oxidative phosphorylation. Electrons stored in the form of the reduced coenzymes, NADH or [FADH 2 ], are passed through an elaborate and highly organized chain of proteins and coenzymes, the so-called electron-transport chain, finally reaching O 2 (molec- ular oxygen), the terminal electron acceptor. Each component of the chain can ex- ist in (at least) two oxidation states, and each component is successively reduced and reoxidized as electrons move through the chain from NADH (or [FADH 2 ]) to O 2 . In the course of electron transport, a proton gradient is established across the inner mitochondrial membrane. It is the energy of this proton gradient that drives ATP synthesis. 20.1 Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur? The processes of electron transport and oxidative phosphorylation are membrane associated. Prokaryotes are the simplest life form, and prokaryotic cells typically consist of a single cellular compartment surrounded by a plasma membrane and a more rigid cell wall. In such a system, the conversion of energy from NADH and [FADH 2 ] to the energy of ATP via electron transport and oxidative phosphorylation is carried out at (and across) the plasma membrane. In eukaryotic cells, electron transport and oxidative phosphorylation are localized in mitochondria, which are also the sites of TCA cycle activity and (as we shall see in Chapter 23) fatty acid oxidation. Mammalian cells contain 800 to 2500 mitochondria; other types of cells may have as few as one or two or as many as half a million mito- chondria. Human erythrocytes, whose purpose is simply to transport oxygen to tis- sues, contain no mitochondria at all. The typical mitochondrion is about 0.5 Ϯ 0.3 mi- cron in diameter and from 0.5 micron to several microns long; its overall shape is sensitive to metabolic conditions in the cell. Mitochondrial Functions Are Localized in Specific Compartments Mitochondria are surrounded by a simple outer membrane and a more complex inner membrane (Figure 20.1). The space between the inner and outer membranes is referred to as the intermembrane space. Several enzymes that utilize ATP (such as creatine kinase and adenylate kinase) are found in the intermembrane space. Wall Piece #IV (1985), a kinetic sculpture by George Rhoads.This complex mechanical art form can be viewed as a metaphor for the molecular apparatus underlying electron transport and ATP synthesis by oxidative phosphorylation. In all things of nature there is something of the marvelous. Aristotle (384–322 B.C.) KEY QUESTIONS 20.1 Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur? 20.2 What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? 20.3 How Is the Electron-Transport Chain Organized? 20.4 What Are the Thermodynamic Implications of Chemiosmotic Coupling? 20.5 How Does a Proton Gradient Drive the Synthesis of ATP? 20.6 What Is the P/O Ratio for Mitochondrial Oxidative Phosphorylation? 20.7 How Are the Electrons of Cytosolic NADH Fed into Electron Transport? 20.8 How Do Mitochondria Mediate Apoptosis? ESSENTIAL QUESTION Living cells save up metabolic energy predominantly in the form of fats and carbohy- drates, and they “spend”this energy for biosynthesis, membrane transport, and move- ment. In both directions, energy is exchanged and transferred in the form of ATP.In Chapters 18 and 19 we saw that glycolysis and the TCA cycle convert some of the en- ergy available from stored and dietary sugars directly to ATP.However,most of the metabolic energy that is obtainable from substrates entering glycolysis and the TCA cycle is funneled via oxidation–reduction reactions into NADH and reduced flavopro- teins, the latter symbolized by [FADH 2 ]. How do cells oxidize NADH and [FADH 2 ] and convert their reducing potential into the chemical energy of ATP? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login.