The Citric Acid Cycle

CHAPTER 16 THE CITRIC ACID CYCLE acetyl groups are oxidized to CO2 in the citric acid cycle, also called the tricarboxylic acid (TCA) cycle or the Krebs cycle (aer its discoverer, Hans Krebs). Much of the energy of these oxidations is conserved in the reduced electron carriers NADH and FADH2. In the third stage of respiration, these reduced coenzymes are themselves oxidized, giving up protons (H+) and electrons. The electrons are transferred to O2 via a series of electron-carrying molecules known as the respiratory chain, resulting in the formation of water. In the course of electron transfer, much of the energy available from redox reactions is conserved in the form of ATP, by a process called oxidative phosphorylation. We discuss this third stage in Chapter 19.

FIGURE 16-1 Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration. Stage 1: oxidation of fatty acids, glucose, and some amino acids yields acetyl-CoA. Stage 2: oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted. Stage 3: electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane–bound) electron carriers — the respiratory chain — ultimately reducing O2 to H2O. This electron flow drives the production of ATP. Hans Krebs, 1900–1981 We use these principles as our guide to this chapter: Pyruvate is the metabolite that links two central catabolic pathways, glycolysis and the citric acid cycle. It is therefore a logical point for regulation that determines the rate of catabolic activity and the partitioning of pyruvate among its possible uses. The reactions of the citric acid cycle follow a chemical logic. In its catabolic role the citric acid cycle oxidizes acetyl- CoA to CO2 and H2O. Energy from the oxidations in the cycle drives the synthesis of ATP. The chemical strategies for activating groups for oxidation and for conserving energy in the form of reducing power and high-energy compounds are used in many other biochemical pathways. The citric acid cycle is a hub of metabolism, with catabolic pathways leading in and anabolic pathways leading out. Acetate groups (acetyl-CoA) from the catabolism of various fuels are used in the synthesis of such metabolites as amino acids, fatty acids, and sterols. The breakdown products of many amino acids and nucleotides are intermediates of the cycle, and they can be fed in or siphoned off as needed by the cell. The central role of the citric acid cycle in metabolism requires that it be regulated in coordination with many other pathways. Regulation occurs by both allosteric and covalent mechanisms that overlap and interact to achieve homeostasis. Some mutations that affect the reactions of the citric acid cycle lead to tumor formation. Enzymes have evolved to form complexes to efficiently achieve a series of chemical transformations without releasing the intermediates into the bulk solvent. This strategy, seen in the pyruvate dehydrogenase complex and the metabolons of the citric acid cycle, is ubiquitous in other pathways of metabolism, in respiration, and in the many “ — somes” that assemble and disassemble informational macromolecules. 16.1 Production of Acetyl-CoA (Activated Acetate) Coenzyme A, or CoA (Fig. 16-2), has a reactive thiol (— SH) group that is critical to its role as an acyl carrier in many metabolic processes, including the citric acid cycle. Acyl groups are covalently linked to the thiol group, forming thioesters. Because of their relatively high standard free energies of hydrolysis (see Figs. 13-16, 13-17), thioesters have a high acyl group transfer potential — that is, donation of their acyl groups to a variety of acceptor molecules is a favorable reaction. The acyl group attached to coenzyme A may thus be thought of as “activated” for group transfer. FIGURE 16-2 Coenzyme A (CoA-SH). A hydroxyl group of pantothenic acid is joined to a modified ADP moiety by a phosphate ester bond, and its carboxyl group is attached to β -mercaptoethylamine in amide linkage. The hydroxyl group at the 3′ position of the ADP moiety has a phosphoryl group not present in free ADP. The — SH group of the mercaptoethylamine moiety forms a thioester with acetate in acetyl-coenzyme A (acetyl- CoA) (lower le ). In aerobic organisms, glucose and other sugars, fatty acids, and most amino acids are ultimately oxidized to CO2 and H2O via the citric acid cycle and the respiratory chain. Before entering the citric acid cycle, the carbon skeletons of monosaccharides and fatty acids are degraded to the acetyl group of acetyl-CoA, the form in which the cycle accepts much of its fuel input. Many amino acid carbons also enter the cycle as acetate, although several amino acids are degraded to other cycle intermediates such as succinate and malate, which then enter the cycle. Pyruvate Is Oxidized to Acetyl-CoA and CO2 Pyruvate generated in the cytosol by glycolysis represents a node in the metabolism of carbohydrates, proteins, and fats. Under anaerobic conditions, pyruvate may simply be reduced to lactate in the cytosol, regenerating NAD+ for continued ATP production by glycolysis. Pyruvate may serve as a precursor for the synthesis of amino acids (Chapter 22). In eukaryotes, pyruvate may diffuse into mitochondria, first through large openings in the outer mitochondrial membrane and then into the matrix via an H+-coupled pyruvate-specific symporter in the inner mitochondrial membrane, the mitochondrial pyruvate carrier (MPC). Pyruvate that enters mitochondria may be oxidized by the citric acid cycle to generate energy or, aer conversion to acetyl- CoA, may be used as the starting material for synthesis of fatty acids and sterols. Pyruvate in the mitochondrial matrix is oxidized to acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex, a highly ordered cluster of enzymes and cofactors. In the PDH complex, a series of chemical intermediates remain bound to the enzyme subunits as a substrate (pyruvate) is transformed into the final product (acetyl-CoA). Five cofactors, four of which are derived from vitamins, participate in the reaction mechanism. The regulation of this enzyme complex illustrates how a combination of covalent modification and allosteric mechanism results in precisely regulated flux through a metabolic step. The overall reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two remaining carbons become the acetyl group of acetyl-CoA (Fig. 16-3). The NADH formed in this reaction gives up a hydride ion (:H−) to the respiratory chain (Fig. 16-1), which carries the two electrons to oxygen or, in anaerobic microorganisms, to an alternative electron acceptor such as nitrate or sulfate. The transfer of electrons from NADH to oxygen ultimately generates 2.5 molecules of ATP per pair of electrons. The irreversibility of the PDH complex reaction has been demonstrated by isotopic labeling experiments: the complex cannot reattach radioactively labeled CO2 to acetyl-CoA to yield carboxyl-labeled pyruvate. FIGURE 16-3 Overall reaction catalyzed by the pyruvate dehydrogenase complex. The five coenzymes participating in this reaction, and the three enzymes that make up the enzyme complex, are discussed in the text. The PDH Complex Employs Three Enzymes and Five Coenzymes to Oxidize Pyruvate The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA (Fig. 16-3) requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups — thiamine pyrophosphate (TPP), lipoate, coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the — SH group), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD). We encountered TPP as the coenzyme of pyruvate decarboxylase (see Fig. 14-13). Lipoate (Fig. 16-4), has two thiol groups that can undergo reversible oxidation to a disulfide bond (— S— S— ), similar to that between two Cys residues in a protein. Because of its capacity to undergo oxidation-reduction reactions, lipoate can serve both as an electron (hydrogen) carrier and as an acyl carrier, as we shall see. CoA serves as the carrier of the activated acyl group. We described the roles of FAD and NAD as electron carriers in Chapter 13. FIGURE 16-4 Lipoic acid (lipoate) in amide linkage with a Lys residue. The lipoyllysyl moiety is the prosthetic group of dihydrolipoyl transacetylase (E2 of the PDH complex). The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol) forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group. The PDH complex contains multiple copies of three enzymes — pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3)—that catalyze the oxidation of pyruvate. The number of copies of each enzyme and therefore the size of the complex varies among species. A central core is formed of many copies (24 to 60) of E2 surrounded by multiple and variable numbers of copies of E1 and E3 (Fig. 16-5). Two regulatory proteins are also part of the complex: a protein kinase and a phosphoprotein phosphatase, discussed below. FIGURE 16-5 Structure of the pyruvate dehydrogenase complex. The complex is so big and so flexible that solving its structure required a combination of methods, including x-ray crystallography and NMR spectroscopy; once the individual pieces were solved, cryo-EM of the whole structure was used to assemble the pieces from several organisms to get this view. The core (E2) is from the gram-negative bacterium Azotobacter vinelandii. E1 and E3 are from the thermophilic gram-positive bacterium Geobacillus stearothermophilus. E1, pyruvate dehydrogenase (yellow); E2, dihydrolipoyl transacetylase (green); and E3, dihydrolipoyl dehydrogenase (red). The central core of the Azotobacter PDH complex consists of 24 copies of E2, but to simplify the structure, only six are shown here. Multiple copies of E1 and E3 surround the central core, and flexible arms (shown schematically) reach out from E2 to E1 and E3, carrying the lipoyl moiety (pink) from the active site of one enzyme to that of the next. The amino acid sequences and three-dimensional structures of individual domains show that both catalytic mechanism and structure have been conserved in evolution. [Information from D. Goodsell, doi:10.2210/rcsb_pdb/mom_2012_9. Data from PDB ID 1LAC F. Dardel et al., J. Mol. Biol. 229:1037, 1993; PDB ID 1EAA A. Mattevi et al., Biochemistry 32:3887, 1993; PDB ID 1W85 R. A. Frank et al., Science 306:872, 2004; PDB ID 1EBD S. S. Mande et al., Structure 4:277, 1996.] The active site of E1 has noncovalently bound TPP. E2 has the prosthetic group lipoate, attached through an amide bond to the ε -amino group of a Lys residue (Fig. 16-4). E2 has three functionally distinct domains: one or more (depending on the species) amino-terminal lipoyl domains, containing the lipoyl-Lys residue(s); the central E1- and E3-binding domain; and the inner- core acyltransferase domain, which contains the acyltransferase active site. The domains of E2 are separated by linkers, sequences of 20 to 30 amino acid residues, rich in Ala and Pro and interspersed with charged residues; these linkers tend to assume their extended forms, holding the three domains apart. The attachment of lipoate to the end of a Lys side chain in E2 produces a long, flexible arm that can move from the active site of E1 to the active sites of E2 and E3, a distance of perhaps 5 nm or more, while holding the intermediate captive throughout the reaction sequence. E3 has tightly bound FAD. This basic E1— E2— E3 structure of the PDH complex is seen in two other enzyme complexes that catalyze similar reactions: α -ketoglutarate dehydrogenase, which oxidizes α - ketoglutarate in the citric acid cycle (described below), and the branched-chain α -keto acid dehydrogenase, which oxidizes α - keto acids derived from the breakdown of the branched-chain amino acids valine, isoleucine, and leucine (see Fig. 18-28). Within a given species, E3 is identical in all three complexes. The remarkable similarity in protein structure, cofactor requirements, and reaction mechanisms of these three complexes doubtless reflects a common evolutionary origin; they are paralogs. The PDH Complex Channels Its Intermediates through Five Reactions Figure 16-6 shows schematically how the pyruvate dehydrogenase complex carries out the five consecutive reactions in the decarboxylation and dehydrogenation of pyruvate without allowing the intermediates to leave the surface of the complex. Step is essentially identical to the reaction catalyzed by pyruvate decarboxylase (see Fig. 14-13c); C-1 of pyruvate is released as CO2, and C-2, which in pyruvate has the oxidation state of an aldehyde, is attached to TPP as a hydroxyethyl group. This first step is the slowest and therefore limits the rate of the overall reaction. In step the hydroxyethyl group is oxidized to the level of a carboxylic acid (acetate). The two electrons removed in this reaction reduce the — S— S— of a lipoyl group on E2 to two thiol (— SH) groups. The acetyl moiety produced in this oxidation- reduction reaction is first esterified to one of the lipoyl — SH groups, then transesterified to CoA to form acetyl-CoA (step ). Thus the energy of oxidation drives the formation of a high- energy thioester of acetate. The remaining reactions catalyzed by the PDH complex (by E3, in steps and ) are electron transfers necessary to regenerate the oxidized (disulfide) form of the lipoyl group of E2, to prepare the enzyme complex for another round of oxidation. The electrons removed from the hydroxyethyl group derived from pyruvate pass through FAD to NAD+, forming NADH, which can enter the respiratory chain. FIGURE 16-6 Oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex. The fate of pyruvate is traced in red. In step pyruvate reacts with the bound thiamine pyrophosphate (TPP) of pyruvate dehydrogenase (E1) and is decarboxylated to the hydroxyethyl derivative. Pyruvate dehydrogenase also carries out step , the transfer of two electrons and the acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core enzyme, dihydrolipoyl transacetylase (E2), to form the acetyl thioester of the reduced lipoyl group. Step is a transesterification in which the — SH group of CoA replaces the — SH group of E2 to yield acetyl-CoA and the fully reduced (dithiol) form of the lipoyl group. In step dihydrolipoyl dehydrogenase (E3) promotes transfer of two hydrogen atoms from the reduced lipoyl groups of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group of E2. In step the reduced FADH2 of E3 transfers a hydride ion to NAD+, forming NADH. The enzyme complex is now ready for another catalytic cycle. (Subunit colors correspond to those in Fig. 16-5.) The five-reaction sequence shown in Figure 16-6 is an example of substrate channeling. The swinging lipoyllysyl arms of E2 accept from E1 the two electrons and the acetyl group that it derived from pyruvate, and pass them to E3. The intermediates of the multistep sequence never leave the complex, and the local concentration of the substrate of E2 is kept very high. This substrate channeling also prevents the of the activated acetyl group by other enzymes that use this group as substrate. We will encounter other enzymes that use a similar tethering mechanism to channel substrate between active sites, with lipoate, biotin, or a CoA-like moiety serving as cofactor. Four different vitamins required in human nutrition are vital components of the pyruvate dehydrogenase complex: thiamine (TPP), pantothenate (CoA), riboflavin (FAD), and niacin (NAD). As one might predict, mutations in the genes for the subunits of the PDH complex, or a dietary vitamin deficiency, can have severe consequences. Thiamine-deficient animals are unable to oxidize pyruvate normally. This is of particular importance to the brain, which usually obtains all its energy from the aerobic oxidation of glucose in a pathway that necessarily includes the oxidation of pyruvate. Beriberi, a disease that results from thiamine deficiency, is characterized by loss of neural function. This disease occurs primarily in populations that rely on a diet consisting mainly of white (polished) rice, which lacks the hulls that contain most of the thiamine found in rice. People who habitually consume large amounts of alcohol can also develop thiamine deficiency, because much of their dietary intake consists of the vitamin-free “empty calories” of distilled spirits. An elevated level of pyruvate in the blood is oen an indicator of defects in pyruvate oxidation due to one of these causes. SUMMARY 16.1 Production of Acetyl- CoA (Activated Acetate) Pyruvate, the end product of glycolysis, enters the mitochondrial matrix, where the pyruvate dehydrogenase (PDH) complex oxidizes it to CO2, acetyl-CoA — the starting material for the citric acid cycle — and NADH. The supramolecular PDH complex includes multiple copies of three enzymes. Pyruvate dehydrogenase, E1 (with bound TPP), decarboxylates pyruvate, producing hydroxyethyl-TPP, which is then oxidized to an acetyl group. The electrons from this oxidation reduce the disulfide of lipoate bound to E2, the dihydrolipoyl transacetylase, and the acetyl group is transferred to one of the — SH groups of the reduced lipoate through a thioester bond. E2 catalyzes the transfer of the acetyl group to coenzyme A, forming acetyl-CoA. Dihydrolipoyl dehydrogenase, E3, catalyzes the regeneration of the disulfide (oxidized) form of lipoate, passing electrons first to FAD, then to NAD+. In the PDH complex we see examples of two strategies that we will see repeated in other metabolic enzyme systems. Its organization is very similar to that of the enzyme complexes that catalyze the oxidation of α -ketoglutarate and the branched-chain α -keto acids. And the long lipoyllysyl arm of E2 is used to channel the substrate from the active site of E1 to E2 to E3, tethering the intermediates to the enzyme complex and increasing the efficiency of the overall reaction. 16.2 Reactions of the Citric AcidCycle We are now ready to trace the process by which acetyl-CoA undergoes oxidation. This chemical transformation is carried out by the citric acid cycle, the first cyclic pathway we have encountered (Fig. 16-7). To begin a turn of the cycle, acetyl-CoA donates its acetyl group to the four-carbon compound oxaloacetate to form the six-carbon citrate. Citrate is then transformed into isocitrate, also a six-carbon molecule, which is dehydrogenated with loss of CO2 to yield the five-carbon compound α -ketoglutarate (also called 2-oxoglutarate). α - Ketoglutarate undergoes loss of a second molecule of CO2 and ultimately yields the four-carbon compound succinate. Succinate is enzymatically converted in three steps into the four-carbon oxaloacetate — which is then ready to react with another molecule of acetyl-CoA. In each turn of the cycle, one acetyl group (two carbons) enters as acetyl-CoA and two molecules of CO2 leave; one molecule of oxaloacetate is used to form citrate and one molecule of oxaloacetate is regenerated. No net removal of oxaloacetate occurs; one molecule of oxaloacetate can theoretically bring about oxidation of an infinite number of acetyl groups, effectively acting catalytically; the steady-state concentration of oxaloacetate is very low (micromolar). Four of the eight steps in this process are oxidations, in which the energy of oxidation is very efficiently conserved in the form of the reduced coenzymes NADH and FADH2. These two carriers donate their electrons to the respiratory chain where electron flow drives ATP synthesis. FIGURE 16-7 Reactions of the citric acid cycle. The carbon atoms shaded in pink are those derived from the acetate of acetyl-CoA in the first turn of the cycle; these are not the carbons released as CO2 in the first turn. Note that in succinate and fumarate, the two-carbon group derived from acetate can no longer be specifically denoted; because succinate and fumarate are symmetric molecules, C-1 and C-2 are indistinguishable from C-4 and C-3. The red arrows show where energy is conserved by electron transfer to FAD or NAD +, forming FADH2 or NADH + H+. Steps , , and are essentially irreversible in the cell; all other steps are reversible. The nucleoside triphosphate product of step may be either ATP or GTP, depending on which succinyl-CoA synthetase isozyme is the catalyst. Although the citric acid cycle is central to energy-yielding metabolism, its role is not limited to energy conservation. Four- and five-carbon intermediates of the cycle serve as precursors for a wide variety of products. To replace intermediates removed for this purpose, cells employ anaplerotic (replenishing) reactions, which are described below. Eugene Kennedy and Albert Lehninger showed in 1948 that, in eukaryotes, the entire set of reactions of the citric acid cycle takes place in mitochondria. Isolated mitochondria were found to contain not only all the enzymes and coenzymes required for the citric acid cycle, but also all the enzymes and proteins necessary for the last stage of respiration — electron transfer and ATP synthesis by oxidative phosphorylation. As we shall see in later chapters, mitochondria also contain the enzymes for the oxidation of fatty acids and some amino acids to acetyl-CoA, and the oxidative degradation of other amino acids to α -ketoglutarate, succinyl-CoA, or oxaloacetate. Thus, in nonphotosynthetic eukaryotes, the mitochondrion is the site of most energy-yielding oxidative reactions and of the coupled synthesis of ATP. In photosynthetic eukaryotes, mitochondria are the major site of ATP production in the dark, but in daylight, chloroplasts produce most of the organism’s ATP. In most bacteria, the enzymes of the citric acid cycle are in the cytosol, and the plasma membrane plays a role analogous to that of the inner mitochondrial membrane in ATP synthesis (Chapter 19). The Sequence of Reactions in the Citric Acid Cycle Makes Chemical Sense Acetyl-CoA produced in the breakdown of carbohydrates, fats, and proteins must be completely oxidized to CO2 if the maximum potential energy is to be extracted from these fuels. However, it is not biochemically feasible to directly oxidize acetate (or acetyl- CoA) to CO2. Decarboxylation of this two-carbon acid would yield CO2 and methane (CH4). Methane is chemically rather stable, and except for certain methanotrophic bacteria that grow in methane-rich niches, organisms do not have the cofactors and enzymes needed to oxidize methane. Methylene groups (— CH2— ), however, are readily metabolized by enzyme systems present in most organisms. In typical oxidation sequences, two adjacent methylene groups (— CH2— CH2— ) are involved, at least one of which is adjacent to a carbonyl group. As we noted in Chapter 13 (p. 473), carbonyl groups are particularly important in the chemical transformations of metabolic pathways. The carbon of the carbonyl group has a partial positive charge due to the electron-withdrawing property of the carbonyl oxygen and is therefore an electrophilic center. A carbonyl group can facilitate the formation of a carbanion on an adjoining carbon by delocalizing the carbanion’s negative charge. We see an example of the oxidation of a methylene group in the citric acid cycle, as succinate is oxidized (steps to in Fig. 16-7) to form a carbonyl (in oxaloacetate) that is more chemically reactive than either a methylene group or methane. In short, if acetyl-CoA is to be oxidized efficiently, the methyl group of the acetyl-CoA must be attached to something. The first step of the citric acid cycle — the condensation of acetyl-CoA with oxaloacetate — neatly solves the problem of the unreactive methyl group. The carbonyl of oxaloacetate acts as an electrophilic center, which is attacked by the methyl carbon of acetyl-CoA in an aldol condensation (p. 473) to form citrate (step in Fig. 16-7). The methyl group of acetate has been converted into a methylene in citric acid. This tricarboxylic acid then readily undergoes a series of oxidations that eliminate two carbons as CO2. Note that all steps featuring the breakage or formation of carbon–carbon bonds (steps , , and ) rely on properly positioned carbonyl groups. As in all metabolic pathways, there is a chemical logic to the sequence of steps in the citric acid cycle: each step either involves an energy-conserving oxidation or is a necessary prelude to the oxidation, placing functional groups in position to facilitate oxidation or oxidative decarboxylation. As you learn the steps of the cycle, keep in mind the chemical rationale for each; it will make the process easier to understand and remember. The Citric Acid Cycle Has Eight Steps In examining the eight successive reaction steps of the citric acid cycle, we place special emphasis on the chemical transformations taking place as citrate formed from acetyl-CoA and oxaloacetate is oxidized to yield CO2 and the energy of this oxidation is conserved in the reduced coenzymes NADH and FADH2. Formation of Citrate The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate synthase. In this reaction, the methyl carbon of the acetyl group is joined to the carbonyl group (C-2) of oxaloacetate. Citroyl-CoA is a transient intermediate formed on the active site of the enzyme (see Fig. 16-9). It undergoes hydrolysis to free CoA and citrate, which are released from the active site. The hydrolysis of this high-energy thioester intermediate makes the forward reaction highly exergonic. The large, negative standard free-energy change of the forward citrate synthase reaction is essential to the operation of the cycle because the concentration of oxaloacetate is normally very low (micromolar). The CoA liberated in this reaction is recycled to participate in the oxidative decarboxylation of another molecule of pyruvate by the PDH complex. Citrate synthase from mitochondria is a homodimer (Fig. 16-8). Each subunit is a single polypeptide with two domains, one large and rigid, the other smaller and more flexible, with the active site between them. Oxaloacetate, the first substrate to bind to the enzyme, induces a large conformational change in the flexible domain, creating a binding site for the second substrate, acetyl-CoA. When citroyl-CoA has formed in the enzyme active site, another conformational change brings about thioester hydrolysis, releasing CoA-SH. This induced fit of the enzyme first to its substrate and then to its reaction intermediate decreases the likelihood of premature and unproductive cleavage of the thioester bond of acetyl-CoA. Kinetic studies of the enzyme are consistent with this ordered bisubstrate mechanism (see Fig. 6-15). The reaction catalyzed by citrate synthase is essentially an aldol condensation (p. 473), involving a thioester (acetyl-CoA) and a ketone (oxaloacetate) (Fig. 16-9).

FIGURE 16-8 Structure of citrate synthase. The flexible domain of each subunit undergoes a large conformational change on binding oxaloacetate, creating a binding site for acetyl-CoA. (a) Open form of the enzyme alone; (b) closed form with bound oxaloacetate and a stable analog of acetyl-CoA (carboxymethyl-CoA). In these representations one subunit is colored tan and one green. [Data from (a) PDB ID 5CSC, D.-I. Liao et al., Biochemistry 30:6031, 1991;] MECHANISM FIGURE 16-9 Citrate synthase. In the citrate synthase reaction in mammals, oxaloacetate binds first, in a strictly ordered reaction sequence. This binding triggers a conformation change that opens up the binding site for acetyl-CoA. Oxaloacetate is specifically oriented in the active site of citrate synthase by interaction of its two carboxylates with two positively charged Arg residues (not shown here). [Information from S. J. Remington, Curr. Opin. Struct. Biol. 2:730, 1992.] Formation of Isocitrate via cis-Aconitate The enzyme aconitase (more formally, aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate, through the intermediary formation of the tricarboxylic acid cis- aconitate, which normally does not dissociate from the active site. Aconitase can promote the reversible addition of H2O to the double bond of enzyme-bound cis-aconitate in two different ways, one leading to citrate and the other to isocitrate: Although the equilibrium mixture at pH 7.4 and 25 °C contains less than 10% isocitrate, in the cell the reaction is pulled to the right because isocitrate is immediately consumed in the next step of the cycle, lowering its steady-state concentration. Aconitase contains an iron-sulfur center (Fig. 16-10), which acts both in the binding of the substrate at the active site and in the catalytic addition or removal of H2O. In iron-depleted cells, acon-itase loses its iron-sulfur center and acquires a new role in the regulation of iron homeostasis. Aconitase is one of many enzymes known to “moonlight” in a second role (Box 16-1). FIGURE 16-10 Iron-sulfur center in aconitase. The iron-sulfur center is in red, the citrate molecule in blue. Three Cys residues of the enzyme bind three iron atoms; the fourth iron is bound to one of the carboxyl groups of citrate and also interacts noncovalently with a hydroxyl group of citrate (dashed bond). A basic residue (:B) in the enzyme helps to position the citrate in the active site. The iron-sulfur center acts in both substrate binding and catalysis. The general properties of iron-sulfur proteins are discussed in Chapter 19. BOX 16-1 Moonlighting Enzymes: Proteins with More Than One Job Before protein and DNA sequencing became routine, two groups of investigators studying two unrelated questions sometimes discovered that “their” proteins had similar properties. Further comparison sometimes showed that they were studying the same protein by following different functions. We now know that many proteins moonlight in a second role, a phenomenon sometimes called gene sharing. Today, researchers using annotated protein and DNA sequence databases can more easily find proteins of the same sequence that have been identified as having different functions, and a protein sequence annotated as having a given function doesn’t necessarily have only that function. When a protein with a known function is made inactive by a mutation, and the resulting mutant organisms show a phenotype with no obvious relation to that function, protein moonlighting can sometimes explain the puzzling result. One citric acid cycle enzyme is a known moonlighter. Eukaryotic cells have two isozymes of aconitase. The mitochondrial isozyme converts citrate to isocitrate in the citric acid cycle. The cytosolic isozyme has two distinct functions. It catalyzes the conversion of citrate to isocitrate, providing the substrate for a cytosolic isocitrate dehydrogenase that generates NADPH as reducing power for fatty acid synthesis and other anabolic processes in the cytosol. But it also has a role in cellular iron homeostasis. All cells must obtain iron for the proteins that require it as a cofactor. In humans, severe iron deficiency results in anemia, an insufficient supply of erythrocytes and a reduced oxygen-carrying capacity that can be life-threatening. Too much iron is also harmful: it accumulates in and damages the liver in hemochromatosis and other diseases. Iron obtained in the diet is carried in the blood by the protein transferrin and enters cells via endocytosis mediated by the transferrin receptor. Once inside cells, iron is used in the synthesis of hemes, cytochromes, Fe-S proteins, and other Fe-dependent proteins; excess iron is stored bound to the protein ferritin. The levels of transferrin, transferrin receptor, and ferritin are therefore crucial to cellular iron homeostasis. The synthesis of these three proteins is regulated in response to iron availability — and aconitase, in its moonlighting job, plays a key regulatory role. Aconitase has an essential Fe-S cluster at its active site (see Fig. 16-10). When a cell is depleted of iron, this Fe-S cluster is disassembled and the enzyme loses its aconitase activity. But the apoenzyme (apoaconitase, lacking its Fe-S cluster) so formed has now acquired its second activity: regulating iron metabolism. Cytosolic apoaconitase is identical to iron regulatory protein 1 (IRP1) and closely related to IRP2. Both IRP1 and IRP2 bind to regions in the mRNAs encoding ferritin and the transferrin receptor, with effects on iron mobilization and iron uptake. These mRNA sequences are part of hairpin structures (see Fig. 8-23) called iron response elements (IREs), located at the 5′ and 3′ ends of the mRNAs (Fig. 1). When bound to the 5′-untranslated IRE sequence in the ferritin mRNA, IRPs block ferritin synthesis; when bound to the 3′-untranslated IRE sequence in the transferrin receptor mRNA, they stabilize the mRNA, preventing its degradation and thus allowing the synthesis of more copies of the receptor protein per mRNA molecule. So, in iron-deficient cells, iron uptake becomes more efficient and iron storage (bound to ferritin) is reduced. When cellular iron concentrations return to normal levels, IRP1 regains its Fe-S cluster, converting to aconitase, and IRP2 undergoes proteolytic degradation, ending the low-iron response. FIGURE 1 Effect of IRP1 and IRP2 on the mRNAs for ferritin and the transferrin receptor. [Information from R. S. Eisenstein, Annu. Rev. Nutr. 20:627, 2000, Fig. 1.] The enzymatically active aconitase and the moonlighting, regulatory apoaconitase have different structures. As the active aconitase, the protein has two lobes that close around the Fe-S cluster; as IRP1, the two lobes open, exposing the mRNA- binding site (Fig. 2). It makes evolutionary sense that the enzymes of central metabolism have had a long time to evolve additional functions. The glycolytic enzyme pyruvate kinase acts in the nucleus to regulate the transcription of genes that respond to thyroid hormone. Glyceraldehyde 3-phosphate dehydrogenase moonlights both as uracil DNA glycosylase, effecting the repair of damaged DNA, and as a regulator of histone H2B transcription. Several glycolytic enzymes, including phosphoglycerate kinase, triose phosphate isomerase, and lactate dehydrogenase, moonlight as crystallins in the lens of the vertebrate eye. FIGURE 2 Two forms of cytosolic aconitase/IRP1 with two distinct functions. (a) In aconitase, the two major lobes are closed and the Fe-S cluster is buried; the protein has been made transparent here to show the Fe-S cluster. (b) In IRP1, the lobes open, exposing a binding site for the mRNA hairpin. [Data from (a) PDB ID 2B3Y, J. Dupuy et al., Structure 14:129, 2006; (b) PDB ID 2IPY, W. E. Walden et al., Science 314:1903, 2006.] Oxidation of Isocitrate to α - Ketoglutarate and CO2 In the next step, isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate to form α -ketoglutarate (Fig. 16-11). M n2+ in the active site interacts with the carbonyl group of the intermediate oxalosuccinate, which is formed transiently but does not leave the binding site until decarboxylation converts it to α - ketoglutarate. M n2+ also stabilizes the enol formed transiently by decarboxylation. MECHANISM FIGURE 16-11 Isocitrate dehydrogenase. In this reaction, the substrate, isocitrate, loses one carbon by oxidative decarboxylation. There are two different forms of isocitrate dehydrogenase in all cells, one requiring NAD + as electron acceptor and the other requiring NADP+. The overall reactions are otherwise identical. In eukaryotic cells, the NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle. The main function of the NADP- dependent enzyme, found in both the mitochondrial matrix and the cytosol, is the generation of NADPH, which is essential for reductive anabolic pathways such as fatty acid and sterol synthesis. Oxidation of α -Ketoglutarate to Succinyl-CoA and CO2 The next step is another oxidative decarboxylation, in which α - ketoglutarate is converted to succinyl-CoA and CO2 by the action of the α -ketoglutarate dehydrogenase complex; NAD + serves as electron acceptor and CoA as the carrier of the succinyl group. The energy of oxidation of α -ketoglutarate is conserved in the formation of the thioester bond of succinyl-CoA: This reaction is virtually identical to the pyruvate dehydrogenase reaction discussed above and to the reaction sequence responsible for the breakdown of branched-chain amino acids (Fig. 16-12). The α -ketoglutarate dehydrogenase complex closely resembles the PDH complex and the complex that degrades branched-chain α -keto acids in both structure and function. All three have homologous E1 and E2 components, identical E3 components, enzyme-bound TPP and lipoate, coenzyme A, FAD, and NAD. These related enzymes can employ the same E3 subunit because the substrate for E3 — a reduced lipoate — is the same for both complexes. They are certainly derived from a common evolutionary ancestor by gene duplication and subsequent divergent evolution, as described in Figure 1-32. FIGURE 16-12 A conserved mechanism for oxidative decarboxylation. The pathways shown employ the same five cofactors (thiamine pyrophosphate, coenzyme A, lipoate, FAD, and NAD +), closely similar multienzyme complexes, and the same enzymatic mechanism to carry out oxidative decarboxylations of pyruvate (by the pyruvate dehydrogenase complex), α -ketoglutarate (in the citric acid cycle), and the carbon skeletons of the three branched-chain amino acids, isoleucine (shown here), leucine, and valine. Conversion of Succinyl-CoA to Succinate Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly negative standard free energy of hydrolysis (ΔG′°≈−36kJ/mol). In the next step of the citric acid cycle, energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in either GTP or ATP, with a net ΔG′° of only –2.9 kJ/mol. Succinate is formed in the process: The enzyme that catalyzes this reversible reaction is called succinyl- CoA synthetase or succinic thiokinase; both names indicate the participation of a nucleoside triphosphate in the reaction. This energy-conserving reaction involves an intermediate step in which the enzyme molecule itself becomes phosphorylated at a His residue in the active site (Fig. 16-13a). This phosphoryl group, which has a high group transfer potential, is transferred to ADP (or GDP) to form ATP (or GTP). Animal cells have two isozymes of succinyl-CoA synthetase, one specific for ADP and the other for GDP. The enzyme has two subunits, α(Mr32,000), which has the –His residue (His246) and the binding site for CoA, and β(Mr42,000), which confers specificity for either ADP or GDP. The active site is at the interface between subunits. The crystal structure of succinyl-CoA synthetase reveals two “power helices” (one from each subunit), oriented so that their electric dipoles situate partial positive charges close to the negatively charged –His (Fig. 16-13b), stabilizing the phosphoenzyme intermediate. (Recall the similar role of helix dipoles in stabilizing K+ ions in the K+ channel; see Fig. 11-45.)

FIGURE 16-13 The succinyl-CoA synthetase reaction. (a) In step a phosphoryl group replaces the CoA of succinyl-CoA bound to the enzyme, forming a high- energy acyl phosphate. In step the succinyl phosphate donates its phosphoryl group to a His residue of the enzyme, forming a high-energy phosphohistidyl enzyme. In step the phosphoryl group is transferred from the His residue to the terminal phosphate of GDP (or ADP), forming GTP (or ATP). (b) Active site of succinyl-CoA synthetase of Escherichia coli. The active site includes part of both the α (blue) and the β (brown) subunits. The power helices (blue, brown) place the partial positive charges of the helix dipole near the phosphate group of -His246 in the α chain, stabilizing the phosphohistidyl enzyme. The bacterial and mammalian enzymes have similar amino acid sequences and three-dimensional structures. [Data from PDB ID 1SCU, W. T. Wolodko et al., J. Biol. Chem. 269:10,883, 1994.] The formation of ATP (or GTP) at the expense of the energy released by the oxidative decarboxylation of α -ketoglutarate is a substrate- level phosphorylation, like the synthesis of ATP in the glycolytic reactions catalyzed by phosphoglycerate kinase and pyruvate kinase (see Fig. 14-2). The GTP formed by succinyl-CoA synthetase can donate its terminal phosphoryl group to ADP to form ATP, in a reversible reaction catalyzed by nucleoside diphosphate kinase (p. 487): G T P + ADP ⇌ G DP + AT P ΔG′°= 0kJ/mol Thus the net result of the activity of either isozyme of succinyl-CoA synthetase is the conservation of energy as ATP. There is no change in free energy for the nucleoside diphosphate kinase reaction; ATP and GTP are energetically equivalent. Oxidation of Succinate to Fumarate The succinate formed from succinyl-CoA is oxidized to fumarate by the flavoprotein succinate dehydrogenase: In eukaryotes, succinate dehydrogenase is an integral protein of the mitochondrial inner membrane; in bacteria, of the plasma membrane. The enzyme contains three different iron-sulfur clusters and one molecule of covalently bound FAD (see Fig. 19-9). Electrons pass from succinate through the FAD and iron-sulfur centers before entering the chain of electron carriers in the mitochondrial inner membrane (the plasma membrane in bacteria). Electron flow from succinate through these carriers to the final electron acceptor, O2, is coupled to the synthesis of about 1.5 ATP molecules per pair of electrons (respiration-linked phosphorylation). Malonate, an analog of succinate not normally present in cells, is a strong competitive inhibitor of succinate dehydrogenase, and its addition to mitochondria in the laboratory blocks the activity of the citric acid cycle. Hydration of Fumarate to Malate The reversible hydration of fumarate to L-malate is catalyzed by fumarase (formally, fumarate hydratase). The transition state in this reaction is a carbanion: This enzyme is highly stereospecific; it catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate. In the reverse direction (from L-malate to fumarate), fumarase is equally stereospecific: D-malate is not a substrate. Oxidation of Malate to Oxaloacetate In the last reaction of the citric acid cycle, L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate, coupled to the reduction of NAD + to NADH:

The equilibrium of this reaction lies far to the le under standard thermodynamic conditions, but in intact cells oxaloacetate is continually removed by the highly exergonic citrate synthase reaction (step of Fig. 16-7). This keeps the concentration of oxaloacetate in the cell extremely low (< 10−6M ), pulling the malate dehydrogenase reaction toward the formation of oxaloacetate. Although the individual reactions of the citric acid cycle were initially worked out in vitro, using minced muscle tissue, the pathway and its regulation have also been studied extensively in vivo. By using precursors isotopically labeled with 14C, researchers have traced the fate of individual carbon atoms through the citric acid cycle. Some of the earliest experiments with 14C produced an unexpected result, however, which aroused considerable controversy about the pathway and mechanism of the citric acid cycle. In fact, these experiments at first seemed to show that citrate was not the first tricarboxylic acid to be formed. Box 16-2 gives some details of this episode in the history of citric acid cycle research. Today biochemists use 13C-labeled precursors and whole-tissue NMR spectroscopy to monitor metabolic flux through the cycle in living tissue. Because the NMR signal is unique to the compound containing the 13C, precursor carbons can be traced into each cycle intermediate and into compounds derived from the intermediates. This technique makes it possible to study regulation of the citric acid cycle and its interconnections with other metabolic pathways. BOX 16-2 Citrate: A Symmetric Molecule That Reacts Asymmetrically When compounds enriched in the heavy-carbon isotope 13C and the radioactive carbon isotopes 11C and 14C became available in the mid-1940s, they were soon put to use in tracing the pathway of carbon atoms through the citric acid cycle. One such experiment initiated the controversy over the role of citrate. Acetate labeled in the carboxyl group (designated [1-14C] acetate) was incubated aerobically with an animal tissue preparation. Acetate is enzymatically converted to acetyl-CoA in animal tissues, and the pathway of the labeled carboxyl carbon of the acetyl group in the cycle reactions could thus be traced. α -Ketoglutarate was isolated from the tissue a er incubation, then degraded by known chemical reactions to establish the position(s) of the isotopic carbon. Condensation of unlabeled oxaloacetate with carboxyl-labeled acetate would be expected to produce citrate labeled in one of the two primary carboxyl groups. Citrate is a symmetric molecule, its two terminal carboxyl groups being chemically indistinguishable. Therefore, half the labeled citrate molecules were expected to yield α -ketoglutarate labeled in the α -carboxyl group and the other half to yield α -ketoglutarate labeled in the γ -carboxyl group; that is, the α -ketoglutarate isolated was expected to be a mixture of the two types of labeled molecules (Fig. 1, pathways and ). Contrary to this expectation, the labeled α -ketoglutarate isolated from the tissue suspension contained 14C only in the γ -carboxyl group (Fig. 1, pathway ). The investigators concluded that citrate (or any other symmetric molecule) could not be an intermediate in the pathway from acetate to α -ketoglutarate. Rather, an asymmetric tricarboxylic acid, presumably cis-aconitate or isocitrate, must be the first product formed from condensation of acetate and oxaloacetate.

FIGURE 1 Incorporation of the isotopic carbon (14C) of the labeled acetyl group into α - ketoglutarate by the citric acid cycle. The carbon atoms of the entering acetyl group are shown in red. In 1948, however, Alexander Ogston pointed out that although citrate has no chiral center (see Fig. 1-19), it has the potential to react asymmetrically if an enzyme with which it interacts has an active site that is asymmetric. He suggested that the active site of aconitase may have three points to which the citrate must be bound and that the citrate must undergo a specific three-point attachment to these binding points. As seen in Figure 2, the binding of citrate to three such points could happen in only one way, and this would account for the formation of only one type of labeled α - ketoglutarate. Organic molecules such as citrate that have no chiral center but are potentially capable of reacting asymmetrically with an asymmetric active site are now called prochiral molecules. FIGURE 2 The prochiral nature of citrate. (a) Structure of citrate; (b) schematic representation of citrate: X = — OH; Y = — COO−; Z = — CH2COO−. (c) Correct complementary fit of citrate to the binding site of aconitase. There is only one way in which the three specified groups of citrate can fit on the three points of the binding site. Thus only one of the two — CH2COO− groups is bound by aconitase. The Energy of Oxidations in the Cycle Is Efficiently Conserved We have now covered one complete turn of the citric acid cycle (Fig. 16-14). A two-carbon acetyl group entered the cycle by combining with oxaloacetate. Two carbon atoms emerged from the cycle as CO2 from the oxidation of isocitrate and α -ketoglutarate. The energy released by these oxidations was conserved in the reduction of three NAD + and one FAD and the production of one ATP or GTP. At the end of the cycle a molecule of oxaloacetate was regenerated. Note that the two carbon atoms appearing as CO2 are not the same two carbons that entered in the form of the acetyl group; additional turns around the cycle are required to release these carbons as CO2 (Fig. 16-7). FIGURE 16-14 Products of one turn of the citric acid cycle. At each turn of the cycle, three NADH, one FADH2, one GTP (or ATP), and two CO2 are released in oxidative decarboxylation reactions. Here and in several of the following figures, all cycle reactions are shown as proceeding in one direction only, but keep in mind that most of the reactions are reversible. Although the citric acid cycle directly generates only one ATP per turn (in the conversion of succinyl-CoA to succinate), the four oxidation steps in the cycle provide a large flow of electrons into the respiratory chain via NADH and FADH2 and thus lead to formation of almost 10 times more ATP during oxidative phosphorylation. We saw in Chapter 14 that the production of two molecules of pyruvate from one molecule of glucose in glycolysis yields 2 ATP and 2 NADH. In oxidative phosphorylation (Chapter 19), passage of two electrons from NADH to O2 drives the formation of about 2.5 ATP, and passage of two electrons from FADH2 to O2 yields about 1.5 ATP. This stoichiometry allows us to calculate the overall yield of ATP from the complete oxidation of glucose. When both pyruvate molecules are oxidized to 6 CO2 via the pyruvate dehydrogenase complex and the citric acid cycle, and the electrons are transferred to O2 via oxidative phosphorylation, as many as 32 ATP are obtained per glucose (Table 16-1). In round numbers, this represents the conservation of 32× 30.5kJ/mol= 976kJ/mol, or 34% of the theoretical maximum of about 2,840 kJ/mol available from the complete oxidation of glucose. These calculations employ the standard free-energy changes; when corrected for the actual free energy required to form ATP within cells (see Worked Example 13-2, p. 480), the calculated efficiency of the process is closer to 65%. When cells under anaerobic conditions depend on glycolysis for ATP, one glucose yields just 2 ATP. Aerobic metabolism is far more effective in capturing the energy in glucose as a fuel. TABLE 16-1 Stoichiometry of Coenzyme Reduction and ATP Formation in the Aerobic Oxidation of Glucose via Glycolysis, the Pyruvate Dehydrogenase Complex Reaction, the Citric Acid Cycle, and Oxidative Phosphorylation Reaction Number of ATP or reduced coenzyme directly formed Number of ATP ultimately formed G lucose →  glucose6-phosphate −1ATP −1 Fructose6-phosphate → fructose1,6-bisphosphate −1ATP −1 2G lyceraldehyde3-phosphate → 21,3-bisphosphoglycerate 2 NADH 3 or 5 21,3-Bisphosphoglycerate → 23-phosphoglycerate 2 ATP 2 2Phosphoenolpyruvate → 2pyruvate 2 ATP 2 2Pyruvate → 2acetyl-CoA 2 NADH 5 2Isocitrate → 2α-ketoglutarate 2 NADH 5 2α-Ketoglutarate → 2succinyl-CoA 2 NADH 5 2Succinyl-CoA → 2succinate 2 ATP (or 2 GTP) 2 2Succinate → 2fumarate 2FADH2 3 2M alate → 2oxaloacetate 2 NADH 5 Total 30–32         This is calculated as 2.5 ATP per NADH and 1.5 ATP per FADH2. A negative value indicates consumption. This number is either 3 or 5, depending on the mechanism used to shuttle NADH equivalents from the cytosol to the mitochondrial matrix; see Figures 19-31 and 19-32. a b a b SUMMARY 16.2 Reactions of the Citric Acid Cycle The citric acid cycle (Krebs cycle, TCA cycle) is a nearly universal central catabolic pathway in which compounds derived from the breakdown of carbohydrates, fats, and proteins are oxidized to CO2, with most of the energy of oxidation temporarily held in the electron carriers FADH2 and NADH. During aerobic metabolism, these electrons are transferred to O2 and the energy of electron flow is conserved as ATP. Acetyl-CoA enters the citric acid cycle as citrate synthase catalyzes its condensation with oxaloacetate to form citrate. In seven sequential reactions, including two decarboxylations, the citric acid cycle converts citrate to oxaloacetate and releases two CO2. The pathway is cyclic in that the intermediates of the cycle are not used up; for each oxaloacetate consumed in the path, one is produced. For each acetyl-CoA oxidized by the citric acid cycle, the energy gain consists of three molecules of NADH, one FADH2, and one nucleoside triphosphate (either ATP or GTP). 16.3 The Hub of Intermediary Metabolism The eight-step cyclic process for oxidation of simple two-carbon acetyl groups to CO2 may seem unnecessarily complex and not in keeping with the biological principle of maximum economy. Remember, though, that the role of the citric acid cycle is not confined to the oxidation of acetate from carbohydrates, fatty acids, or amino acids. The cycle also accepts 3-, 4-, and 5- carbon skeletons, especially from the breakdown of amino acids, at other points in the pathway. For example, when deaminated, the amino acids aspartate and glutamate become the cycle intermediates oxaloacetate and α -ketoglutarate, respectively. The Citric Acid Cycle Serves in Both Catabolic and Anabolic Processes In aerobic organisms, the citric acid cycle is an amphibolic pathway, one that serves in both catabolic and anabolic processes. In the cycle’s anabolic role, oxaloacetate and α - ketoglutarate can be withdrawn from the cycle to serve as precursors of aspartate and glutamate by simple transamination (Chapter 22). Through aspartate and glutamate, the carbons of oxaloacetate and α -ketoglutarate are then used to build other amino acids, as well as purine and pyrimidine nucleotides. Succinyl-CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups, which serve as oxygen carriers (in hemoglobin and myoglobin) and electron carriers (in cytochromes) (see Figs. 22-25, 22-26). Finally, oxaloacetate can be converted to glucose via gluconeogenesis (see Fig. 14-16). One biosynthetic process is not possible for animals: the conversion of acetate or acetyl-CoA to glucose. Given that the carbon atoms of acetate molecules entering the citric acid cycle appear eight steps later in oxaloacetate, it might seem that the cycle could generate oxaloacetate from acetate, then the oxaloacetate could be used to synthesize glucose by gluconeogenesis. However, there is no net conversion of acetate to oxaloacetate; for every two carbons that enter the cycle as acetate (acetyl-CoA), two leave as CO2. In bacteria, plants, fungi, and protists, another reaction sequence, the glyoxylate cycle, serves as a mechanism for converting acetate to carbohydrate. The glyoxylate cycle, which shares some reactions with the citric acid cycle, converts two molecules of acetate to one of oxaloacetate in a variant of the citric acid cycle in which the two decarboxylation steps are bypassed (see Fig. 20-45). Thus, plants and many simpler organisms can synthesize glucose from fatty acids, but humans and other animals cannot. Anaplerotic Reactions Replenish Citric Acid Cycle Intermediates Under most circumstances, there is a dynamic steady state between reactions that siphon intermediates away from the citric acid cycle and those that supply additional carbon skeletons. When the withdrawal of cycle intermediates for use in biosynthesis lowers the concentrations of citric acid cycle intermediates enough to slow the cycle, the intermediates are replenished by anaplerotic reactions (Greek, “to refill”) (Fig. 16- 15). Table 16-2 shows the most common anaplerotic reactions, all of which, in various tissues and organisms, convert either pyruvate or phosphoenolpyruvate to oxaloacetate or malate. The most important anaplerotic reaction in mammalian liver, kidney, and brown adipose tissue is the reversible carboxylation of pyruvate by HCO− 3 to form oxaloacetate, catalyzed by pyruvate carboxylase. The enzymatic addition of a carboxyl group to pyruvate requires energy, which is supplied by ATP.

FIGURE 16-15 Role of the citric acid cycle in anabolism. Intermediates of the citric acid cycle are drawn off as precursors in many biosynthetic pathways. Shown in red are four anaplerotic reactions that replenish depleted cycle intermediates (see Table 16-2). TABLE 16-2 Anaplerotic Reactions Reaction Tissue(s)/organism(s) Liver, kidney Heart, skeletal muscle Higher plants, yeast, bacteria Widely distributed in eukaryotes and bacteria Pyruvate carboxylase is a regulatory enzyme and is virtually inactive in the absence of acetyl-CoA, its positive allosteric modulator. Whenever acetyl-CoA, the fuel for the citric acid cycle, is present in excess, it stimulates the pyruvate carboxylase reaction to produce more oxaloacetate, enabling the cycle to use more acetyl-CoA in the citrate synthase reaction. Biotin in Pyruvate Carboxylase Carries One-Carbon (CO2) Groups The pyruvate carboxylase reaction requires the vitamin biotin (Fig. 16-16), which is the prosthetic group of the enzyme. Biotin, which plays a key role in many carboxylation reactions, is a specialized carrier of one-carbon groups in their most oxidized form: CO2. (The transfer of one-carbon groups in more reduced forms is mediated by other cofactors, notably tetrahydrofolate and S-adenosylmethionine, as described in Chapter 18.) Carboxyl groups are activated in a reaction that consumes ATP and joins CO2 to enzyme-bound biotin. This “activated” CO2 is then passed to an acceptor (pyruvate in this case) in a carboxylation reaction. MECHANISM FIGURE 16-16 The role of biotin in the reaction catalyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the ε -amino group of a Lys residue, forming biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed in separate active sites on the enzyme, as exemplified by the pyruvate carboxylase reaction. In the first phase (steps to ), bicarbonate is converted to the more activated CO2, and then used to carboxylate biotin. The biotin acts as a carrier to transport the CO2 from one active site to another on an adjacent monomer of the tetrameric enzyme (step ). In the second phase (steps to ), catalyzed in this second active site, the CO2 reacts with pyruvate to form oxaloacetate. Pyruvate carboxylase has four identical subunits, each containing a molecule of biotin covalently attached through an amide linkage to the ε -amino group of a specific Lys residue in the enzyme active site. Carboxylation of pyruvate proceeds in two steps (Fig. 16-16): first, a carboxyl group derived from HCO− 3 is attached to biotin, then the carboxyl group is transferred to pyruvate to form oxaloacetate. These two steps occur at separate active sites; the long flexible arm of biotin transfers activated carboxyl groups from the first active site (on one monomer of the tetramer) to the second (on the adjacent monomer), functioning much like the long lipoyllysyl arm of E2 in the PDH complex (Fig. 16-6) and the long arm of the CoA-like moiety in the acyl carrier protein involved in fatty acid synthesis (see Fig. 21-5); these are compared in Figure 16-17. Lipoate, biotin, and pantothenate all enter cells on the same transporter; all become covalently attached to proteins by similar reactions; and all provide a flexible tether that allows bound reaction intermediates to move from one active site to another in an enzyme complex, without dissociating from it. That is, all participate in substrate channeling. FIGURE 16-17 Biological tethers. The cofactors lipoate, biotin, and the combination of β -mercaptoethylamine and pantothenate form long, flexible arms (green) on the enzymes to which they are covalently bound, acting as tethers that move intermediates from one active site to the next. The group shaded light red is, in each case, the point of attachment of the activated intermediate to the tether. SUMMARY 16.3 The Hub of Intermediary Metabolism The citric acid cycle is amphibolic, serving in both catabolism and anabolism. Besides acetyl-CoA, any compound that gives rise to a four- or five-carbon intermediate of the citric acid cycle — for example, the breakdown products of many amino acids — can be oxidized by the cycle. Cycle intermediates can be drawn off and used as the starting material for a variety of biosynthetic products. When intermediates are shunted from the citric acid cycle to other pathways, they are replenished by several anaplerotic reactions, which produce four-carbon intermediates by carboxylation of three-carbon compounds; pyruvate carboxylase is a major anaplerotic enzyme. Enzymes that catalyze carboxylations commonly employ biotin to activate CO2 and to carry it to acceptors such as pyruvate or phosphoenolpyruvate. 16.4 Regulation of the Citric Acid Cycle The central role of the citric acid cycle in metabolism requires stringent regulation to balance the supply of key intermediates with the demands of energy production and biosynthetic processes. Regulation occurs at several levels, including the oxidation of pyruvate to acetyl-CoA (catalyzed by the PDH complex) and the entry of acetyl-CoA into the cycle (the citrate synthase reaction). The transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier (MPC) determines to some degree the fate of pyruvate produced by glycolysis. Most cells also produce acetyl-CoA from the oxidation of fatty acids (Chapter 17) and certain amino acids (Chapter 18), and the availability of intermediates from those other pathways is important in the regulation of pyruvate oxidation and of the citric acid cycle. There are therefore multiple points at which the metabolism of pyruvate and the citric acid cycle may be regulated. Production of Acetyl-CoA by the PDH Complex Is Regulated by Allosteric and Covalent Mechanisms The PDH complex of mammals is strongly inhibited allosterically by ATP and by acetyl-CoA and NADH, the products of the reaction catalyzed by the complex (Fig. 16-18). Long-chain fatty acids, which can be broken down to acetyl-CoA, are also inhibitory. AMP, CoA, and NAD +, all of which accumulate when too little acetate flows into the citric acid cycle, allosterically activate the PDH complex. Thus, PDH complex activity is turned off when ample fuel is available in the form of fatty acids and acetyl- CoA and when the cell’s [ATP]/[AD P] and [NAD H]/[NAD +] ratios are high; it is turned on again when energy demands are high and the cell requires greater flux of acetyl-CoA into the citric acid cycle. FIGURE 16-18 Regulation of metabolite flow from the PDH complex through the citric acid cycle in mammals. The PDH complex is allosterically inhibited when [ATP]/[AD P], [NAD H]/[NAD +], and [acetyl-CoA]/[CoA] ratios are high, all of which indicate an energy- sufficient metabolic state. When these ratios decrease, allosteric activation of pyruvate oxidation results. The rate of flow through the citric acid cycle can be limited by the availability of the citrate synthase substrates, oxaloacetate and acetyl-CoA, or of NAD +, which is depleted by its conversion to NADH, slowing the three NAD +-dependent oxidation steps. Feedback inhibition by succinyl-CoA, citrate, and ATP also slows the cycle by inhibiting early steps. In muscle tissue, Ca2+ stimulates contraction and, as shown here, stimulates energy-yielding metabolism to replace the ATP consumed by contraction. In mammals, these allosteric regulatory mechanisms are complemented by a second level of regulation: phosphorylation/dephosphorylation. The PDH complex is inhibited by reversible phosphorylation of Ser residues on E1 by PDH kinase, which is an intrinsic part of the PDH complex. PDH kinase is allosterically activated by the products of the PDH complex (ATP, NADH, and acetyl-CoA), and it is inhibited by the substrates for the PDH complex (ADP, NAD +, and pyruvate) (Fig. 16-19). The complex also contains PDH phosphatase, which reverses the inhibition by PDH kinase. Together, the kinase and phosphatase exert strong control over the entry of acetyl-CoA from pyruvate into the citric acid cycle. Higher concentrations of ATP, NADH, or acetyl-CoA lead to inactivation of the PDH complex by phosphorylation of PDH. When the concentrations of ADP, NAD +, or pyruvate rise, kinase activity decreases and pyruvate dehydrogenase phosphatase removes the phosphoryl group, reactivating the PDH complex and thereby stimulating the citric acid cycle. FIGURE 16-19 Pyruvate dehydrogenase is inactivated by phosphorylation catalyzed by pyruvate dehydrogenase kinase. The kinase is regulated by metabolites that signal the energetic state of the cell. Metabolites that accumulate in an energy-sufficient state activate PDH kinase, which phosphorylates and inactivates PDH. Pyruvate is then diverted away from the energy-yielding citric acid cycle (CAC). Metabolites indicating energy need or pyruvate accumulation have the opposite effect, keeping PDH active and sending acetyl-CoA into the CAC. The simple compound dichloroacetate (DCA), a structural analog of acetate, inhibits PDH kinase in the laboratory and so relieves the inhibition of the PDH complex. This stimulates pyruvate oxidation via the citric acid cycle (Fig. 16-19) and thus may have a use in directing metabolism in tumor cells away from aerobic glycolysis (see Box 14-1). The increase in mitochondrial oxidation also stimulates apoptosis (Chapter 19), thereby suppressing tumor growth. Phase III clinical trials of DCA began in 2019. The Citric Acid Cycle Is Also Regulated at Three Exergonic Steps Each of the three strongly exergonic steps in the cycle — those catalyzed by citrate synthase, isocitrate dehydrogenase, and α - ketoglutarate dehydrogenase (Fig. 16-18) — can become the rate- limiting step under some circumstances. The availability of the substrates for citrate synthase (acetyl-CoA and oxaloacetate) varies with the metabolic state of the cell and sometimes limits the rate of citrate formation. NADH, a product of isocitrate and α -ketoglutarate oxidation, accumulates under some conditions, and at high [NAD H]/[NAD +] both dehydrogenase reactions are severely inhibited by mass action. Similarly, the malate dehydrogenase reaction is essentially at equilibrium in the cell (that is, it is substrate-limited), and when [NAD H]/[NAD +] is high the concentration of oxaloacetate is low, slowing the first step in the cycle. Product accumulation inhibits all three limiting steps of the cycle: succinyl-CoA inhibits α -ketoglutarate dehydrogenase (and also citrate synthase); citrate blocks citrate synthase; and the end product, ATP, inhibits both citrate synthase and isocitrate dehydrogenase. The inhibition of citrate synthase by ATP is relieved by ADP, an allosteric activator of this enzyme. In vertebrate muscle, Ca2+, the signal for contraction and for a concomitant increase in demand for ATP, activates both isocitrate dehydrogenase and α -ketoglutarate dehydrogenase, as well as the PDH complex. In short, the concentrations of substrates and intermediates in the citric acid cycle set the flux through this pathway at a rate that provides optimal concentrations of ATP and NADH. Under normal conditions, the rates of glycolysis and of the citric acid cycle are integrated so that only as much glucose is metabolized to pyruvate as is needed to supply the citric acid cycle with fuel (acetyl-CoA). Both pathways are inhibited by high levels of ATP and NADH, but also by the concentration of citrate, the product of the first step of the citric acid cycle, and an important allosteric inhibitor of phosphofructokinase-1 of the glycolytic pathway (see Fig. 14-23). Citric Acid Cycle Activity Changes in Tumors The mitochondrial pyruvate carrier (MPC) is down-regulated in tumor cells, leading to pyruvate accumulation in the cytosol. Several other mitochondrial enzymes are also inactivated in tumor cells, including the PDH complex and succinate dehydrogenase. As a result, tumor cells accumulate lactate (from the pyruvate produced by glycolysis) and succinate. Both of these intermediates are oncometabolites; they stimulate tumor growth, acting through specific G protein–coupled receptors (GPCRs; see Chapter 12) in the plasma membrane. The membrane receptor for lactate is upregulated in most cancers. L- Lactate acting through its membrane receptor, lowers [cAMP] and raises [Ca2+], with downstream effects currently under investigation. Mutations in citric acid cycle enzymes are very rare in humans and other mammals, but those that do occur are devastating. Genetic defects in succinate dehydrogenase lead to tumors of the adrenal gland (pheochromocytomas), and mutations in the fumarase gene lead to tumors of smooth muscle (leiomyomas) and kidney. Their activities thus define both enzymes as tumor suppressors (p. 451). Another remarkable connection between citric acid cycle intermediates and cancer is the finding that in many glial cell tumors (gliomas), the NADPH-dependent isocitrate dehydrogenase has an unusual genetic defect. The mutant enzyme loses its normal activity (converting isocitrate to α - ketoglutarate) but gains a new activity: it converts α -ketoglutarate to 2-hydroxyglutarate (Fig. 16-20), which accumulates in the tumor cells. α -Ketoglutarate and Fe3+ are essential cofactors for a family of histone demethylases that alter gene expression. They do so by removing methyl groups from Arg and Lys residues in the histones that organize nuclear DNA. By competing with α - ketoglutarate for binding to the histone demethylases, 2- hydroxyglutarate inhibits their activity. Inhibition of the histone demethylases interferes with normal gene regulation, leading to unrestricted glial cell growth. The family of more than 60 dioxygenases that use α -ketoglutarate and Fe3+ as cofactors are also competitively inhibited by 2-hydroxyglutarate. Inhibition of one or more of these enzymes could interfere with normal regulation of cell division and thus produce a tumor.

FIGURE 16-20 A mutant isocitrate dehydrogenase acquires a new activity. Wild-type isocitrate dehydrogenase catalyzes the conversion of isocitrate to α -ketoglutarate, but mutations that alter the binding site for isocitrate cause loss of the normal enzymatic activity and gain of a new activity: conversion of α -ketoglutarate to 2-hydroxyglutarate. Accumulation of this product inhibits histone demethylase, altering gene regulation and leading to glial cell tumors in the brain. Certain Intermediates Are Channeled through Metabolons We saw an example of substrate channeling in the five-reaction sequence of the PDH complex. Many other reactions occur in similar multienzyme complexes that ensure efficient passage of the product of one enzyme reaction to the next enzyme in the pathway. Such integrated multienzyme complexes, metabolons, are held together by noncovalent interactions and are not easily extracted intact from the cell. In the classic approach of enzymology — purification of individual proteins from extracts of broken cells — once cells are broken open, their contents, including enzymes, are diluted 100- or 1,000-fold (Fig. 16-21), which favors dissociation of noncovalent complexes such as metabolons.

FIGURE 16-21 Effect of protein concentration on complex stability. Dilution of a solution containing a noncovalently bound protein complex — such as a metabolon consisting of three enzymes (illustrated here in red, blue, and green) — favors dissociation of the complex into its constituents. The enzymes of the citric acid cycle are usually described as soluble components of the mitochondrial matrix (except for succinate dehydrogenase, which is membrane-bound), but there is evidence that at least three sequential enzymes of the citric acid cycle (malate dehydrogenase, citrate synthase, and aconitase) constitute a metabolon (Fig. 16-22). We will see more examples of substrate channeling in some amino acid biosynthesis pathways in Chapter 22. As cryo-EM increases our understanding of larger complexes and “ — somes” (apoptosomes, respirasomes, and replisomes, for example), it seems likely that many more enzymes once thought to function as individual soluble proteins will be found to act in multienzyme complexes that facilitate the channeling of substrates. FIGURE 16-22 A three-enzyme metabolon of the citric acid cycle. (a) Purified porcine enzymes malate dehydrogenase (MDH), citrate synthase (CS), and aconitase form a metabolon when combined in vitro. (b) Electrostatic modeling shows that a broad path of positive potential along the surface of a MDH-CS complex connects the active sites of MDH and CS. This path provides a channel for the passage of the negatively charged oxaloacetate (OAA) from the active site of MDH, where it is formed from - malate, to the active site of CS, where it condenses with acetyl-CoA to form citrate. Engineered mutations that replaced a positively charged Arg residue along this path with a negatively charged Asp residue greatly reduced the rate of substrate channeling through the complex, providing evidence that the functional unit is a metabolon. [Information from B. Bulutoglu et al., ACS Chem. Biol. 11:2847, 2016. Data from PDB ID 1MLD, W. B. Gleason et al., Biochemistry 33:2078, 1994; PDB ID 1CTS, S. Remington et al., J. Mol. Biol. 158:111, 1982; PDB ID 7ACN, H. Lauble et al., Biochemistry 31:2735, 1992.] SUMMARY 16.4 Regulation of the Citric Acid Cycle The production of acetyl-CoA for the citric acid cycle by the PDH complex is inhibited allosterically by metabolites that signal a sufficiency of metabolic energy (ATP, acetyl-CoA, NADH, and fatty acids) and is stimulated by metabolites that indicate a reduced energy supply (AMP, CoA-SH, NAD +). The PDH complex is regulated by allosteric mechanisms and covalent modification (phosphorylation). The overall rate of the citric acid cycle is controlled by the rate of conversion of pyruvate to acetyl-CoA and by the flux through citrate synthase, isocitrate dehydrogenase, and α -ketoglutarate dehydrogenase. These fluxes are affected by the concentrations of substrates and products: the end products ATP and NADH are inhibitory, and the substrates NAD + and ADP are stimulatory. Long-chain fatty acids, which can break down to acetyl-CoA, are also inhibitory. Some mutations that affect the PDH complex or citric acid cycle enzymes are oncogenic: they occur very commonly in certain types of cancer. Complexes of consecutive enzymes in a pathway (metabolons) allow substrate channeling and more efficient passage of substrates through the reaction sequences. Chapter Review Key Terms Terms in bold are defined in the glossary. cellular respiration citric acid cycle tricarboxylic acid (TCA) cycle Krebs cycle thioester mitchondrial pyruvate carrier (MPC) pyruvate dehydrogenase (PDH) complex oxidative decarboxylation lipoate substrate channeling iron-sulfur center α -ketoglutarate dehydrogenase complex moonlighting enzymes nucleoside diphosphate kinase prochiral molecule amphibolic pathway glyoxylate cycle anaplerotic reaction biotin oncometabolite metabolon PROBLEMS 1. Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, α -ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. a. Write a balanced equation for the reaction catalyzed by each enzyme. b. Name the cofactor(s) required by each enzyme reaction. c. For each enzyme, determine which of the following describes the type of reaction(s) catalyzed: condensation (carbon–carbon bond formation); dehydration (loss of water); hydration (addition of water); decarboxylation (loss of CO2); oxidation- reduction; substrate-level phosphorylation; isomerization. d. Write a balanced net equation for the catabolism of acetyl-CoA to CO2. 2. Net Equation for Glycolysis and the Citric Acid Cycle Write the net biochemical equation for the metabolism of a molecule of glucose by glycolysis and the citric acid cycle, including all cofactors. 3. Recognizing Oxidation and Reduction Reactions One biochemical strategy of many living organisms is the stepwise oxidation of organic compounds to CO2 and H2O and the conservation of a major part of the energy thus produced in the form of ATP. It is important to be able to recognize oxidation-reduction processes in metabolism. Reduction of an organic molecule results from the hydrogenation of a double bond (Eqn 1, below) or of a single bond with accompanying cleavage (Eqn 2). Conversely, oxidation results from dehydrogenation. In biochemical redox reactions, the coenzymes NAD and FAD dehydrogenate/hydrogenate organic molecules in the presence of the proper enzymes.

For each of the metabolic transformations (a) through (h), determine whether the compound on the le has undergone oxidation or reduction. Balance each transformation by inserting H— H and, where necessary, H2O. a. b. c. d. e.

f. g. h. 4. Relationship between Energy Release and the Oxidation State of Carbon A eukaryotic cell can use glucose (C6H12O6) and hexanoate (C6H11O2) as fuels for cellular respiration. On the basis of their structural formulas, which substance releases more energy per gram on complete combustion to CO2 and H2O? 5. Nicotinamide Coenzymes as Reversible Redox Carriers The nicotinamide coenzymes (see Fig. 13-24) can undergo reversible oxidation-reduction reactions with specific substrates in the presence of the appropriate dehydrogenase. In these reactions, NAD H + H+ serves as the hydrogen source, as described in Problem 3. Whenever the coenzyme is oxidized, a substrate must be simultaneously reduced: SubstrateOxidized + NAD H Reduced + H+ ⇌ product Reduced + NAD + Oxidized For each of the reactions in (a) through (f) shown below, determine whether the substrate has been oxidized or reduced or is unchanged in oxidation state (see Problem 3). If a redox change has occurred, balance the reaction with the necessary amount of NAD +, NADH, H+, and H2O. The objective is to recognize when a redox coenzyme is necessary in a metabolic reaction. a. b.

c. d. e. f. 6. Pyruvate Dehydrogenase Cofactors and Mechanism Describe the role of each cofactor involved in the reaction catalyzed by the pyruvate dehydrogenase complex. 7. Thiamine Deficiency Individuals with a thiamine-deficient diet have relatively high levels of pyruvate in their blood. Explain this in biochemical terms. 8. Isocitrate Dehydrogenase Reaction What type of chemical reaction is involved in the conversion of isocitrate to α - ketoglutarate? Name and describe the role of any cofactors. What other reaction(s) of the citric acid cycle are of this same type? 9. Stimulation of Oxygen Consumption by Oxaloacetate and Malate In the early 1930s, Albert Szent-Györgyi reported the interesting observation that the addition of small amounts of oxaloacetate or malate to suspensions of minced pigeon breast muscle stimulated the oxygen consumption of the preparation. Surprisingly, the amount of oxygen consumed was about seven times more than the amount necessary for complete oxidation (to CO2 and H2O) of the added oxaloacetate or malate. Why did the addition of oxaloacetate or malate stimulate oxygen consumption? Why was the amount of oxygen consumed so much greater than the amount necessary to completely oxidize the added oxaloacetate or malate? 10. Formation of Oxaloacetate in a Mitochondrion In the last reaction of the citric acid cycle, malate is dehydrogenated to regenerate the oxaloacetate necessary for the entry of acetyl-CoA into the cycle: L-M alate+ NAD + →  oxaloacetate+ NAD H + H+ ΔG′°= 30.0kJ /mol a. Calculate the equilibrium constant for this reaction at 25 °C. b. Because ΔG′° assumes a standard pH of 7, the equilibrium constant calculated in (a) corresponds to K′eq = The measured concentration of L-malate in rat liver mitochondria is about 0.20 mM when [NAD +]/[NAD H] is 10. Calculate the concentration of oxaloacetate at pH 7 in these mitochondria. c. To appreciate the magnitude of the mitochondrial oxaloacetate concentration, calculate the number of oxaloacetate molecules in a single rat liver mitochondrion. Assume the mitochondrion is a sphere of diameter 2.0μm. 11. Cofactors for the Citric Acid Cycle Suppose you have prepared a mitochondrial extract that contains all the soluble enzymes of the matrix but has lost (by dialysis) all the low molecular weight cofactors. What must you add to the extract so that the preparation will oxidize acetyl-CoA to CO2? 12. Riboflavin Deficiency How would a riboflavin deficiency affect the functioning of the citric acid cycle? Explain your answer. 13. Oxaloacetate Pool What factors might decrease the pool of oxaloacetate available for the activity of the citric acid cycle? How can the pool of oxaloacetate be replenished? [oxaloacetate][NAD H] [L-malate][NAD +] 14. Energy Yield from the Citric Acid Cycle The reaction catalyzed by succinyl-CoA synthetase produces the high- energy compound GTP. How is the free energy contained in GTP incorporated into the cellular ATP pool? 15. Respiration Studies in Isolated Mitochondria Cellular respiration can be studied in isolated mitochondria by measuring oxygen consumption under different conditions. If 0.01 M sodium malonate is added to actively respiring mitochondria that are using pyruvate as fuel, respiration soon stops and a metabolic intermediate accumulates. a. What is the structure of this intermediate? b. Explain why it accumulates. c. Explain why oxygen consumption stops. d. Aside from removal of the malonate, what can overcome this inhibition of respiration? Explain. 16. Labeling Studies in Isolated Mitochondria Biochemists have oen delineated the metabolic pathways of organic compounds by using a radioactively labeled substrate and following the fate of the label. a. How can you determine whether a suspension of isolated mitochondria metabolizes added glucose to CO2 and H2O? b. Suppose you add a brief pulse of [3-14C]pyruvate (labeled in the methyl position) to the mitochondria. Aer one turn of the citric acid cycle, what is the location of the 14C in the oxaloacetate? Explain by tracing the 14C label through the pathway. How many turns of the cycle are required to release all the [3-14C]pyruvate as CO2? 17. [1-14C]GlucoseCatabolism An investigator briefly incubates an actively respiring bacterial culture with [1-14C]glucose and isolates the glycolytic and citric acid cycle intermediates. Where is the 14C located in each of the intermediates listed? Consider only the initial incorporation of 14C in the first pass of labeled glucose through the pathways. a. Fructose 1,6-bisphosphate b. Glyceraldehyde 3-phosphate c. Phosphoenolpyruvate d. Acetyl-CoA e. Citrate f. α -Ketoglutarate g. Oxaloacetate 18. Role of the Vitamin Thiamine People with beriberi, a disease caused by thiamine deficiency, have elevated levels of blood pyruvate and α -ketoglutarate, especially aer consuming a meal rich in glucose. How are these effects related to a deficiency of thiamine? 19. Synthesis of Oxaloacetate by the Citric Acid Cycle In the last step of the citric acid cycle, NAD +-dependent oxidation of L-malate forms oxaloacetate. Can a net synthesis of oxaloacetate from acetyl-CoA occur using only the enzymes and cofactors of the citric acid cycle, without depleting the intermediates of the cycle? Explain. How do cells replenish the oxaloacetate that is lost from the cycle to biosynthetic reactions? 20. Oxaloacetate Depletion Mammalian liver can carry out gluconeogenesis using oxaloacetate as the starting material (Chapter 14). Would the extensive use of oxaloacetate for gluconeogenesis affect the operation of the citric acid cycle? Explain your answer. 21. Mode of Action of the Rodenticide Fluoroacetate Fluoroacetate, prepared commercially for rodent control, is also produced by a South African plant. Aer entering a cell, fluoroacetate is converted to fluoroacetyl-CoA in a reaction catalyzed by the enzyme acetate thiokinase: You perform a perfusion experiment to study the toxic effect of fluoroacetate using intact isolated rat heart. Aer perfusing the heart with 0.22 mM fluoroacetate, you see a decrease in the measured rate of glucose uptake and glycolysis as well as an accumulation of glucose 6-phosphate and fructose 6-phosphate. Examination of the citric acid cycle intermediates reveals that their concentrations are below normal, except for citrate, which has a concentration 10 times higher than normal. a. Where did the block in the citric acid cycle occur? What caused citrate to accumulate and the other cycle intermediates to be depleted? b. Fluoroacetyl-CoA is enzymatically transformed in the citric acid cycle. What is the structure of the end product of fluoroacetate metabolism? Why does it block the citric acid cycle? How might the inhibition be overcome? c. In the heart perfusion experiments, why did glucose uptake and glycolysis decrease? Why did hexose monophosphates accumulate? d. Why is fluoroacetate poisoning fatal? 22. Synthesis of L-Malate in Wine Making The tartness of some wines is due to high concentrations of L-malate. Write a sequence of reactions showing how yeast cells synthesize L- malate from glucose under anaerobic conditions in the presence of dissolved CO2(HCO− 3). Note that the overall reaction for this fermentation cannot involve the consumption of nicotinamide coenzymes or citric acid cycle intermediates. 23. Net Synthesis of α -Ketoglutarate α -Ketoglutarate plays a central role in the biosynthesis of several amino acids. Write a sequence of enzymatic reactions that could result in the net synthesis of α -ketoglutarate from pyruvate. Your proposed sequence must not involve the net consumption of other citric acid cycle intermediates. Write an equation for the overall reaction. 24. Amphibolic Pathways Explain, giving examples, what is meant by the statement that the citric acid cycle is amphibolic. 25. Regulation of the Pyruvate Dehydrogenase Complex In animal tissues, the ratio of active, unphosphorylated to inactive, phosphorylated PDH complex regulates the rate of conversion of pyruvate to acetyl-CoA. Determine what happens to the rate of this reaction when a preparation of rabbit muscle mitochondria containing the PDH complex is treated with (a) pyruvate dehydrogenase kinase, ATP, and NADH; (b) pyruvate dehydrogenase phosphatase and Ca2+; (c) malonate. 26. Commercial Synthesis of Citric Acid Manufacturers use citric acid as a flavoring agent in so drinks, fruit juices, and many other foods. Worldwide, the market for citric acid is valued at hundreds of millions of dollars per year. Commercial production uses the mold Aspergillus niger, which metabolizes sucrose under carefully controlled conditions. a. The yield of citric acid strongly depends on the concentration of FeCl3 in the culture medium, as indicated in the graph. Why does the yield decrease when the concentration of Fe3+ is above or below the optimal value of 0.5 mg/L? b. Write the sequence of reactions by which A. niger synthesizes citric acid from sucrose. Write an equation for the overall reaction. c. Does the commercial process require the culture medium to be aerated — that is, is this a fermentation or an aerobic process? Explain. 27. Regulation of Citrate Synthase In the presence of saturating amounts of oxaloacetate, the activity of citrate synthase from pig heart tissue shows a sigmoid dependence on the concentration of acetyl-CoA, as shown in the graph. Adding succinyl-CoA shis the curve to the right and makes the sigmoid dependence more pronounced. On the basis of these observations, suggest how succinyl-CoA regulates the activity of citrate synthase. (Hint: See Fig. 6-37.) Why is succinyl-CoA an appropriate signal for regulation of the citric acid cycle? How does the regulation of citrate synthase control the rate of cellular respiration in pig heart tissue? 28. Regulation of Pyruvate Carboxylase The carboxylation of pyruvate by pyruvate carboxylase occurs at a very low rate unless acetyl-CoA, a positive allosteric modulator, is present. If you have just eaten a meal rich in fatty acids (triacylglycerols) but low in carbohydrates (glucose), how does this regulatory property shut down the oxidation of glucose to CO2 and H2O but increase the oxidation of acetyl- CoA derived from fatty acids? 29. Relationship between Respiration and the Citric Acid Cycle Although oxygen does not participate directly in the citric acid cycle, the cycle operates only when O2 is present. Why? 30. Effect of [NAD H]/[NAD +] on the Citric Acid Cycle How would you expect the operation of the citric acid cycle to respond to a rapid increase in the [NAD H]/[NAD +] ratio in the mitochondrial matrix? Why? 31. Thermodynamics of Citrate Synthase Reaction in Cells Citrate is formed by the condensation of acetyl-CoA with oxaloacetate, catalyzed by citrate synthase: Oxaloacetate+ acetyl-CoA + H2O ⇌  citrate+ CoA + H+ In rat heart mitochondria at pH 7.0 and 25 °C, the concentrations of reactants and products are oxaloacetate, 1μM ; acetyl-CoA, 1μM ; citrate, 220μM ; and CoA, 65μM . The standard free-energy change for the citrate synthase reaction is −32.2kJ /mol. What is the direction of metabolite flow through the citrate synthase reaction in rat heart cells? Explain. 32. Reactions of the Pyruvate Dehydrogenase Complex Two of the steps in the oxidative decarboxylation of pyruvate (steps and in Fig. 16-6) do not involve any of the three carbons of pyruvate, yet are essential to the operation of the PDH complex. Explain. 33. Pyruvate Transport into Mitochondria The mitochondrial pyruvate carrier (MPC) is a heterodimer of the proteins MPC1 and MPC2. In a high proportion (80%) of certain cancers, including gliomas (tumors of the glial cells of the brain), the gene for one of these proteins is mutated such that pyruvate cannot enter the mitochondrial matrix. Name three metabolic effects that you would expect to see if cytosolic pyruvate could not gain access to the machinery of the citric acid cycle. (Hint: Box 14-1 may be helpful.) 34. Citric Acid Cycle Mutants There are many cases of human disease in which one or another enzyme activity is lacking due to genetic mutation. Why are cases in which individuals lack one of the enzymes of the citric acid cycle extremely rare? DATA ANALYSIS PROBLEM 35. How the Citric Acid Cycle Was Discovered The detailed biochemistry of the citric acid cycle was determined by several researchers over a period of decades. In a 1937 article, Krebs and Johnson summarized their work and the work of others in the first published description of this pathway. The methods used by these researchers were very different from those of modern biochemistry. Radioactive tracers were not commonly available until the 1940s, so Krebs and other researchers had to use nontracer techniques to work out the pathway. Using freshly prepared samples of pigeon breast muscle, they determined oxygen consumption by suspending minced muscle in buffer in a sealed flask and measuring the volume (in μ L) of oxygen consumed under different conditions. They measured levels of substrates (intermediates) by treating samples with acid to remove contaminating proteins, then assaying the quantities of various small organic molecules. The two key observations that led Krebs and colleagues to propose a citric acid cycle as opposed to a linear pathway (like that of glycolysis) were made in the following experiments. Experiment I: They incubated 460 mg of minced muscle in 3 mL of buffer at 40 °C for 150 minutes. Addition of citrate increased O2 consumption by 893 μ L compared with samples without added citrate. They calculated, based on the O2 consumed during respiration of other carbon-containing compounds, that the expected O2 consumption for complete respiration of this quantity of citrate was only 302 μ L. Experiment II: They measured O2 consumption by 460 mg of minced muscle in 3 mL of buffer when incubated with citrate and/or with 1-phosphoglycerol (glycerol 1-phosphate; this was known to be readily oxidized by cellular respiration) at 40 °C for 140 minutes. The results are shown in the table. Sample Substrate(s) added μLO2 absorbed 1 No extra    342 2 0.3 mL 0.2 1-phosphoglycerol    757 3 0.15 mL 0.02 citrate    431 4 0.3 mL 0.2 1-phosphoglycerol and 0.15 mL 0.02 citrate 1,385 a. Why is O2 consumption a good measure of cellular respiration? b. Why does sample 1 (unsupplemented muscle tissue) consume some oxygen? c. Based on the results for samples 2 and 3, can you conclude that 1-phosphoglycerol and citrate serve as substrates for cellular respiration in this system? Explain your reasoning. d. Krebs and colleagues used the results from these experiments to argue that citrate was “catalytic” — that it helped the muscle tissue samples metabolize 1- phosphoglycerol more completely. How would you use their data to make this argument? e. Krebs and colleagues further argued that citrate was not simply consumed by these reactions, but had to be regenerated. Therefore, the reactions had to be a cycle rather than a linear pathway. How would you make this argument? Other researchers had found that arsenate (AsO3− 4 ) inhibits α -ketoglutarate dehydrogenase and that malonate inhibits succinate dehydrogenase. f. Krebs and coworkers found that muscle tissue samples treated with arsenate and citrate would consume citrate only in the presence of oxygen; under these conditions, oxygen was consumed. Based on the pathway in Figure 16-7, what was the citrate converted to in this experiment, and why did the samples consume oxygen? In their article, Krebs and Johnson further reported the following: (1) In the presence of arsenate, 5.48 mmol of citrate was converted to 5.07 mmol of α - ketoglutarate. (2) In the presence of malonate, citrate was quantitatively converted to large amounts of succinate and small amounts of α -ketoglutarate. (3) Addition of oxaloacetate in the absence of oxygen led to production of a large amount of citrate; the amount was increased if glucose was also added. Other workers had found the following pathway in similar muscle tissue preparations: g. Based only on the data presented in this problem, what is the order of the intermediates in the citric acid cycle? How does this compare with Figure 16-7? Explain your reasoning. Succinate →  fumarate →  malate →  oxaloacetate −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ p h. Why was it important to show the quantitative conversion of citrate to α -ketoglutarate? The Krebs and Johnson article also contains other data that filled in most of the missing components of the cycle. The only component le unresolved was the molecule that reacted with oxaloacetate to form citrate. Reference Krebs, H.A., and W.A. Johnson. 1937. The role of citric acid in intermediate metabolism in animal tissues. Enzymologia 4:148–156. Reprinted in FEBS Lett. 117(Suppl.):K2–K10, 1980.