Biochemistry, 4th Edition P61 docx

© Richard Cummins/CORBIS 19 The Tricarboxylic Acid Cycle Under aerobic conditions, pyruvate from glycolysis is converted to acetyl-coenzyme A (acetyl-CoA) and oxidized to CO 2 in the tricarboxylic acid (TCA) cycle (also called the citric acid cycle). The electrons liberated by this oxidative process are passed via NADH and FADH 2 through an elaborate, membrane-associated electron- transport pathway to O 2 , the final electron acceptor. Electron transfer is coupled to creation of a proton gradient across the membrane. Such a gradient represents an energized state, and the energy stored in this gradient is used to drive the synthesis of many equivalents of ATP. ATP synthesis as a consequence of electron transport is termed oxidative phosphorylation; the complete process is diagrammed in Figure 19.1. Aerobic pathways permit the production of 30 to 38 molecules of ATP per glucose oxidized. Although two molecules of ATP come from glycolysis and two more directly out of the TCA cycle, most of the ATP arises from oxidative phosphorylation. Specifically, reducing equivalents released in the oxidative reactions of glycolysis, pyruvate de- carboxylation, and the TCA cycle are captured in the form of NADH and enzyme- bound FADH 2 , and these reduced coenzymes fuel the electron-transport pathway and oxidative phosphorylation. Complete oxidation of glucose to CO 2 involves the removal of 24 electrons—that is, it is a 24-electron oxidation. In glycolysis, 4 electrons are removed as NADH, and 4 more exit as two more NADH in the decarboxylation of two molecules of pyruvate to two acetyl-CoA (Figure 19.1). For each acetyl-CoA oxidized in the TCA cycle, 8 more electrons are removed (as three NADH and one FADH 2 ): H 3 CCOO Ϫ ϩ 2H 2 O ϩ H ϩ ⎯⎯→ 2CO 2 ϩ 8H In the electron-transport pathway these 8 electrons combine with oxygen to form water: 8H ϩ 2O 2 ⎯⎯→ 4H 2 O So, the net reaction for the TCA cycle and electron transport pathway is H 3 CCOO Ϫ ϩ 2O 2 ϩ H ϩ ⎯⎯→ 2CO 2 ϩ 2H 2 O As German biochemist Hans Krebs showed in the 1930s, the eight-electron oxi- dation of acetate by the TCA cycle is accomplished with the help of oxaloacetate. (In his honor, the TCA cycle is often referred to as the Krebs cycle.) Beginning with acetate, a series of five reactions produces two molecules of CO 2 , with four electrons extracted in the form of NADH and four electrons passed to oxaloacetate to pro- duce a molecule of succinate. The pathway becomes a cycle by three additional re- actions that accomplish a four-electron oxidation of succinate back to oxaloacetate. This special trio of reactions is used repeatedly in metabolism: first, oxidation of a single bond to a double bond, then addition of the elements of water across the double bond, and finally A time-lapse photograph of a ferris wheel at night. Aerobic cells use a metabolic wheel—the tricarboxylic acid cycle—to generate energy by acetyl-CoA oxidation. Thus times do shift, each thing his turn does hold; New things succeed, as former things grow old. Robert Herrick Hesperides (1648), “Ceremonies for Christmas Eve” KEY QUESTIONS 19.1 What Is the Chemical Logic of the TCA Cycle? 19.2 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA? 19.3 How Are Two CO 2 Molecules Produced from Acetyl-CoA? 19.4 How Is Oxaloacetate Regenerated to Complete the TCA Cycle? 19.5 What Are the Energetic Consequences of the TCA Cycle? 19.6 Can the TCA Cycle Provide Intermediates for Biosynthesis? 19.7 What Are the Anaplerotic, or “Filling Up,” Reactions? 19.8 How Is the TCA Cycle Regulated? 19.9 Can Any Organisms Use Acetate as Their Sole Carbon Source? ESSENTIAL QUESTION 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 glucose. Under anaerobic conditions, pyruvate is reduced to lactate in animals and to ethanol in yeast, and much of the potential energy of the glucose molecule remains untapped. In the presence of oxygen, however, a much more interesting and thermodynamically complete story unfolds. How is pyruvate oxidized under aerobic conditions, and what is the chemical logic that dictates how this process occurs? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. 564 Chapter 19 The Tricarboxylic Acid Cycle oxidation of the resulting alcohol to a carbonyl. We will see it again in fatty acid oxidation (see Chapter 23), in reverse in fatty acid synthesis (see Chapter 24), and in amino acid synthesis and breakdown (see Chapter 25). 19.1 What Is the Chemical Logic of the TCA Cycle? The entry of new carbon units into the cycle is through acetyl-CoA. This entry metabolite can be formed either from pyruvate (from glycolysis) or from oxidation of fatty acids (discussed in Chapter 23). Transfer of the two-carbon acetyl group from acetyl-CoA to the four-carbon oxaloacetate to yield six-carbon citrate is cat- alyzed by citrate synthase. A dehydration–rehydration rearrangement of citrate yields isocitrate. Two successive decarboxylations produce ␣-ketoglutarate and then succinyl-CoA, a CoA conjugate of a four-carbon unit. Several steps later, oxaloac- etate is regenerated and can combine with another two-carbon unit of acetyl-CoA. Thus, carbon enters the cycle as acetyl-CoA and exits as CO 2 . In the process, meta- bolic energy is captured in the form of ATP, NADH, and enzyme-bound FADH 2 (symbolized as [FADH 2 ]). The TCA Cycle Provides a Chemically Feasible Way of Cleaving a Two-Carbon Compound The cycle shown in Figure 19.2 at first appears to be a complicated way to oxidize acetate units to CO 2 , but there is a chemical basis for the apparent complexity. Oxidation of an acetyl group to a pair of CO 2 molecules requires COC cleavage: CH 3 COO Ϫ ⎯⎯→CO 2 ϩ CO 2 O 2 H 2 O Acetyl-CoA Citric acid cycle ␣-Ketoglutarate Isocitrate Citrate Succinyl-CoA Fumarate Oxaloacetate Malate Succinate [FADH 2 ] FADH 2 + Electron transport Oxidative phosphorylation Proton gradient Mitochondrial matrix Intermembrane space + H + H + H + H + H + H + H + H + H + H + H + e – e – e – GTP GDP P i P i NADH NADH NADH NADH ADP ATP e – Pyruvate Glucose Glycolysis NAD + NADH NAD + NADH (a) (b) (c) ATP ADP + P i FIGURE 19.1 (a) Pyruvate produced in glycolysis is oxidized in (b) the tricar- boxylic acid (TCA) cycle.(c) Electrons liberated in this oxidation flow through the electron-transport chain and drive the synthesis of ATP in oxidative phosphory- lation. In eukaryotic cells, this overall process occurs in mitochondria. 19.1 What Is the Chemical Logic of the TCA Cycle? 565 1 Pyruvate dehydrogenase Citrate synthase 2 Aconitase 3 Isocitrate dehydrogenase ␣-Ketoglutarate dehydrogenase 4 5 Nucleoside diphosphate kinase Succinyl-CoA synthetase Succinate dehydrogenase Fumarase 6 7 8 Malate dehydrogenase Isocitrate H 2 C COO – HC COO – HC OH COO – C O Acetyl-CoA CS O Pyruvate CH 3 C H 3 C H 2 C C O O – O From glycolysis From ␤-oxidation of fatty acids Oxaloacetate COO – COO – COO – COO – H 2 C COO – H 2 C COO – H 2 C COO – HO C Citrate ␣-Ketoglutarate C C SCoA Succinyl-CoA O O Succinate FADH 2 FAD C C Fumarate H H HC HO H 2 C COO – H 2 C H 2 C COO – H 2 C H 2 C COO – COO – H 2 C COO – Malate TRICARBOXYLIC ACID CYCLE (citric acid cycle, Krebs cycle, TCA cycle) H 2 O H 2 O COO – – OOC ATP GTP GDP P i ADP CoASH NADH + NAD + NAD + NAD + NAD + CoASH CoASH CO 2 CoA NADH + NADH + NADH + H + CO 2 CO 2 H + H + H + ACTIVE FIGURE 19.2 The TCA cycle. Test yourself on the concepts in this figure at www.cengage.com/login. 566 Chapter 19 The Tricarboxylic Acid Cycle In many instances, COC cleavage reactions in biological systems occur between car- bon atoms ␣ and ␤ to a carbonyl group: A good example of such a cleavage is the fructose bisphosphate aldolase reaction (see Figure 18.12). Another common type of COC cleavage is ␣-cleavage of an ␣-hydroxyketone: (We see this type of cleavage in the transketolase reaction described in Chapter 22.) Neither of these cleavage strategies is suitable for acetate. It has no ␤-carbon, and the second method would require hydroxylation—not a favorable reaction for ac- etate. Instead, living things have evolved the clever chemistry of condensing acetate with oxaloacetate and then carrying out a ␤-cleavage. The TCA cycle combines this ␤-cleavage reaction with oxidation to form CO 2 , regenerate oxaloacetate, and cap- ture the liberated metabolic energy in NADH and ATP. 19.2 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA? Pyruvate produced by glycolysis is a significant source of acetyl-CoA for the TCA cy- cle. Because, in eukaryotic cells, glycolysis occurs in the cytoplasm, whereas the TCA cycle reactions and all subsequent steps of aerobic metabolism take place in the mi- tochondria, pyruvate must first enter the mitochondria to enter the TCA cycle. The oxidative decarboxylation of pyruvate to acetyl-CoA Pyruvate ϩ CoA ϩ NAD ϩ ⎯⎯→acetyl-CoA ϩ CO 2 ϩ NADH is the connecting link between glycolysis and the TCA cycle. The reaction is cat- alyzed by pyruvate dehydrogenase, a multienzyme complex. The pyruvate dehydrogenase complex (PDC) is formed from multiple copies of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). All are involved in the conversion of pyru- vate to acetyl-CoA. The active sites of all three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme, and so on, without diffusion of substrates and products through the solution. Eukaryotic PDC, one of the largest-known multienzyme complexes (with a di- ameter of approximately 500 Å) is a 9.5-megadalton assembly organized around an icosahedral 60-mer of E2 subunits, with 30 E1 heterotetramers and 12 homodimers of E3 (Figure 19.3). Eukaryotic PDC also contains an E3-binding protein (E3BP) that is required to bind E3 to the PDC. Trimeric units of E2 form the 20 vertices of the icosahedron, with E3BP bound in each of the 12 faces. The E2 subunits each carry a lipoic acid moiety covalently linked to a lysine residue. Flexible linker seg- ments in E2 and E3BP impart the flexibility that allows the lipoic acid groups to visit all three active sites during catalysis. The pyruvate dehydrogenase reaction (Figure 19.4) is a tour de force of mecha- nistic chemistry, involving as it does a total of three enzymes and five different coen- zymes. The first step of this reaction, decarboxylation of pyruvate and transfer of the C OOH C ␣ Cleavage C O C ␣ C ␤ Cleavage 19.2 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA? 567 acetyl group to lipoic acid, depends on accumulation of negative charge on the transferred two-carbon fragment, as facilitated by the quaternary nitrogen on the thiazolium group of thiamine pyrophosphate (TPP). As shown in Figure 19.5, this cationic imine nitrogen plays two distinct roles in TPP-catalyzed reactions: 1. It provides electrostatic stabilization of the thiazole carbanion formed upon re- moval of the C-2 proton. (The sp 2 hybridization and the availability of vacant d orbitals on the adjacent sulfur probably also facilitate proton removal at C-2.) 2. TPP attack on pyruvate leads to decarboxylation. The TPP cationic imine nitro- gen can act as an effective electron sink to stabilize the negative charge that must develop on the carbon that has been attacked. This stabilization takes place by resonance interaction through the double bond to the nitrogen atom. This resonance-stabilized intermediate can be protonated to give hydroxyethyl- TPP. The reaction of hydroxyethyl-TPP with the oxidized form of lipoic acid yields the energy-rich acetyl-thiol ester of reduced lipoic acid through oxidation of the hydroxyl-carbon of the two-carbon substrate unit. Nucleophilic attack by CoA on the carbonyl carbon (a characteristic feature of CoA chemistry) results in transfer of the acetyl group from lipoic acid to CoA. The subsequent oxidation of lipoic acid is catalyzed by the FAD-dependent dihydrolipoyl dehydrogenase, and NAD ϩ is reduced. FIGURE 19.3 Icosahedral model of PDC core structure (E3 not shown). E1 subunits (yellow) are joined to the E2 core (green) by linkers (blue). (Adapted from Zhou, Z. H., McCarthy, D. B., O’Connor, C. M., Reed, L. J., and Stoops, J. K., 2001. The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes. Proc Natl Acad Sci U S A 98:14802–14807. Figure courtesy of Z. Hong Zhou.) CH 3 C O CH 3 COO – C O CH 3 CH OH TPP CH 3 C O [FAD] + H + SCoA Protein SH SH S H S S S CoASH NAD + NADH Pyruvate Thiamine pyrophosphate Hydroxyethyl TPP (HETPP) Pyruvate loses CO 2 and HETPP is formed Hydroxyethyl group is transferred to lipoic acid and oxidized to form acetyl dihydrolipoate Acetyl group is transferred to CoA Lipoic acid is reoxidized Pyruvate dehydrogenase Dihydrolipoyl transacetylase (dihydrolipoamide acetyltransferase) Dihydrolipoyl dehydrogenase (dihydrolipoamide dehydrogenase) Lipoic acid CO 2 3 12 4 3 123 4 FIGURE 19.4 The reaction mechanism of the pyruvate dehydrogenase complex.Decarboxylation of pyruvate occurs with formation of hydroxyethyl-TPP (step 1).Transfer of the two-carbon unit to lipoic acid in step 2 is fol- lowed by formation of acetyl-CoA in step 3. Lipoic acid is reoxidized in step 4 of the reaction. 568 Chapter 19 The Tricarboxylic Acid Cycle A DEEPER LOOK The Coenzymes of the Pyruvate Dehydrogenase Complex Coenzymes are small molecules that bring unique chemistry to en- zyme reactions. Five coenzymes are used in the pyruvate dehydro- genase reaction. Thiamine Pyrophosphate TPP assists in the decarboxylation of ␣-keto acids (here) and in the formation and cleavage of ␣-hydroxy ketones (as in the trans- ketolase reaction, see Chapter 22). NH 2 N NH 3 C H C H N + H 3 C H C H H C H OH + NH 2 N N H 3 C H C H H C H H C H O O O – P O O – P O O – S N + S H 3 C ATP H H Thiamine (vitamin B 1 ) TPP Synthetase Thiamine pyrophosphate (TPP) Acidic proton AMP O C O N NH 2 + H O HH OHOH H CH 2 P – O P O – OOCH 2 H O HH OHOH H N N N N NH 2 O C O N NH 2 HH H 1 2 3 4 6 5 O Nicotinamide (oxidized form) Nicotinamide (reduced form) Hydride ion, H – pro-R pro-S AMP NADP + contains a P i on this 2Ј-hydroxyl Nicotinamide adenine dinucleotide, NAD + The Nicotinamide Coenzymes NAD ϩ /NADH and NADP ϩ /NADPH carry out hydride (HϺ Ϫ ) transfer reactions. All reactions involving these coenzymes are two-electron transfers. 19.2 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA? 569 H 3 C N H H 3 C N N N O CH 2 HCOH CH 2 O P O O – O P O O – O CH 2 O OH OH HH HH N N N N NH 2 D-Ribitol O HCOH HCOH H 3 C N H 3 C N N NH O R 8 7 6 9 9a 5a 10 4a 5 4 3 2 1 10a H + , e – H + , e – H 3 C N H 3 C N N NH O R H O H 3 C N H 3 C N N N O R O H pK a ≅ 8.4 H 3 C N H 3 C N N N O R H O H H O H + , e – H + , e – – H – + H + H – + H + Isoalloxazine AMP Riboflavin Flavin mononucleotide, FMN Flavin adenine dinucleotide, FAD FAD or FMN FADH or FMNH Oxidized form ␭ max = 450 nm (yellow) Semiquinone form ␭ max = 570 nm (blue) Semiquinone anion ␭ max = 490 nm (red) FADH 2 or FMNH 2 Reduced form (colorless) (a) (b) The Flavin Coenzymes—FAD/FADH 2 Flavin coenzymes can exist in any of three redox states, and this allows flavin coenzymes to participate in one-electron transfer and two-electron transfer reactions. Partly because of this, flavoproteins catalyze many reactions in biological systems and work with many electron acceptors and donors. Because the ribityl group is not a true pentose sugar (it is a sugar alcohol) and is not joined to riboflavin in a glyco- sidic bond, the molecule is not truly a “nucleotide” and the terms flavin mononucleotide and dinucleotide are incorrect. Nonetheless, these designations are so deeply ingrained in common biochemical usage that the erroneous nomencla- ture persists. Continued 570 Chapter 19 The Tricarboxylic Acid Cycle A DEEPER LOOK The Coenzymes of the Pyruvate Dehydrogenase Complex (cont’d) Coenzyme A The two main functions of CoA are: 1. Activation of acyl groups for transfer by nucleophilic attack 2. Activation of the ␣-hydrogen of the acyl group for abstraction as a proton The reactive sulfhydryl group on CoA mediates both of these func- tions. The sulfhydryl group forms thioester linkages with acyl groups. The two main functions of CoA are illustrated in the citrate synthase reaction (see Figure 19.6). β OC CH 2 SH CH 2 NH OC CH 2 CH 2 NH HCOH H 3 C C CH 3 CH 2 O PO – O O PO – O O CH 2 O HH OOH HH PO 3 2 – N N N N NH 2 3' -Mercaptoethylamine Pantothenic acid 3؅,5؅–ADP 4-Phosphopantetheine S S CHCH 2 CH 2 CH 2 CH 2 CH 2 C CH 2 O – O S S O N CH HN O H C HS HS H 2 C CH 2 CHCH 2 CH 2 CH 2 CH 2 C O – O (a) (b) (c) Lipoic acid Lysine Lipoic acid, oxidized form Reduced form Lipoyllysine (lipoamide) Lipoic Acid Lipoic acid functions to couple acyl-group transfer and electron trans- fer during oxidation and decarboxylation of ␣-keto acids. It is found in pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase. Lipoic acid is covalently bound to relevant enzymes through amide bond for- mation with the ⑀-NH 2 group of a lysine side chain. 19.3 How Are Two CO 2 Molecules Produced from Acetyl-CoA? 571 19.3 How Are Two CO 2 Molecules Produced from Acetyl-CoA? The Citrate Synthase Reaction Initiates the TCA Cycle The first reaction within the TCA cycle, the one by which carbon atoms are intro- duced, is the citrate synthase reaction (Figure 19.6). Here acetyl-CoA reacts with ox- aloacetate in a Perkin condensation (a carbon–carbon condensation between a ketone or aldehyde and an ester). The acyl group is activated in two ways in an acyl-CoA mol- ecule: The carbonyl carbon is activated for attack by nucleophiles, and the C ␣ carbon is more acidic and can be deprotonated to form a carbanion. The citrate synthase re- action depends upon the latter mode of activation. As shown in Figure 19.6, 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. This part of the reaction has an equilibrium constant near 1, but the overall reaction is driven to completion by the subsequent Li p oamide C CH 3 COO – O S N R" R R' B + S N R" R R' + BH S N R" R R' + C CH 3 C OH OO S N R" R R' + C CH 3 OH S N R" R R' C CH 3 OH H + H + H + S N R" R R' C CH 3 OH H E E H – – – : B C CH 3 O H S S Lipoamide H : B C CH 3 CH 3 O O H S SH _ : B C CH 3 O CoA S C CoA S H : + CO 2 – – : N N N Lipoamide S SH Lipoamide S SH Lipoamide SHSH Resonance-stabilized carbanion on substrate Hydroxyethyl-TPP Pyruvate FIGURE 19.5 The mechanistic details of the first three steps of the pyruvate dehydrogenase complex reaction. O C C SCoA H H O B B H H C COO – COO – Oxaloacetate H 2 C H 2 O H 2 C C SCoA O HO C COO – H 2 C COO – Citryl-CoA H 2 C HO C COO – H 2 C COO – COO – Citrate pro-S arm pro-R arm + E CoA E FIGURE 19.6 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA.The mechanism involves nucleophilic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis. Go to CengageNOW and click CengageInteractive to explore the citrate synthase reaction. 572 Chapter 19 The Tricarboxylic Acid Cycle hydrolysis of the high-energy thioester to citrate and free CoA. The overall ⌬G°Ј is Ϫ31.4 kJ/mol, and under standard conditions the reaction is essentially irreversible. Although the mitochondrial concentration of oxaloacetate is very low (much less than 1 ␮M—see example in Section 19.4), the strong, negative ⌬G°Ј drives the reaction forward. Citrate Synthase Is a Dimer Citrate synthase in mammals is a dimer of 49-kD sub- units (Table 19.1). On each subunit, oxaloacetate and acetyl-CoA bind to the active site, which lies in a cleft between two domains and is surrounded mainly by ␣-helical segments (Figure 19.7). Binding of oxaloacetate induces a conformational change that facilitates the binding of acetyl-CoA and closes the active site so that the reactive carbanion of acetyl-CoA is protected from protonation by water. NADH Is an Allosteric Inhibitor of Citrate Synthase Citrate synthase is the first step in this metabolic pathway, and as stated the reaction has a large negative ⌬G°Ј. As might be expected, it is a highly regulated enzyme. NADH, a product of the TCA cycle, is an allosteric inhibitor of citrate synthase, as is succinyl-CoA, the product of the fifth step in the cycle (and an acetyl-CoA analog). Citrate Is Isomerized by Aconitase to Form Isocitrate Citrate itself poses a problem: It is a poor candidate for further oxidation because it contains a tertiary alcohol, which could be oxidized only by breaking a carbon–carbon bond. An obvious solution to this problem is to isomerize the ter- tiary alcohol to a secondary alcohol, which the cycle proceeds to do in the next step. Citrate is isomerized to isocitrate by aconitase in a two-step process involving aconitate as an intermediate (Figure 19.8). In this reaction, 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 ef- fect is the conversion of a tertiary alcohol (citrate) to a secondary alcohol (iso- citrate). Oxidation of the secondary alcohol of isocitrate involves breakage of a COH bond, a simpler matter than the COC cleavage required for the direct oxi- dation of citrate. Inspection of the citrate structure shows a total of four chemically equivalent hy- drogens, but only one of these—the pro-R H atom of the pro-R arm of citrate—is ab- stracted by aconitase, which is quite stereospecific. Formation of the double bond of aconitate following proton abstraction requires departure of hydroxide ion from the C-3 position. Hydroxide is a relatively poor leaving group, and its departure is facili- tated in the aconitase reaction by coordination with an iron atom in an iron–sulfur cluster. ⌬G°؅ ⌬G Reaction Enzyme (kJ/mol) (kJ/mol) 1. Acetyl-CoA ϩ oxaloacetate ϩ H 2 O 34 CoASH ϩ citrate Citrate synthase Ϫ31.4 Ϫ53.9 2. Citrate 34 isocitrate Aconitase ϩ6.7 ϩ0.8 3. Isocitrate ϩ NAD ϩ 34 ␣-ketoglutarate ϩ NADH ϩ CO 2 Isocitrate dehydrogenase Ϫ8.4 Ϫ17.5 4. ␣-Ketoglutarate ϩ CoASH ϩ NAD ϩ 34 succinyl-CoA ϩ NADH ϩ CO 2 ␣-Ketoglutarate Ϫ30 Ϫ43.9 dehydrogenase complex 5. Succinyl-CoA ϩ GDP ϩ P i 34 succinate ϩ GTP ϩ CoASH Succinyl-CoA synthetase Ϫ3.3 Ϸ0 6. Succinate ϩ [FAD] 34 fumarate ϩ [FADH 2 ] Succinate dehydrogenase ϩ0.4 0 7. Fumarate ϩ H 2 O 34 L-malate Fumarase Ϫ3.8 Ϸ0 8. L-Malate ϩ NAD ϩ 34 oxaloacetate ϩ NADH ϩ H ϩ Malate dehydrogenase ϩ29.7 Ϸ0 ⌬G values from Newsholme, E. A.,and Leech, A. R.,1983. Biochemistry for the Medical Sciences. New York: Wiley. TABLE 19.1 The Enzymes and Reactions of the TCA Cycle CoASH FIGURE 19.7 Citrate synthase.In the monomer shown here, citrate is shown in blue, and CoA is red.(Top: pdb id ϭ 1CTS; bottom:pdb id ϭ 2CTS.)