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SECTION 8 The Citric Acid Cycle Learning Objectives ✓ Why is the reaction catalyzed by the pyruvate dehydrogenase complex a crucial juncture in metabolism? ✓ How is the pyruvate dehydrogenase complex regulated? ✓ What is the advantage of oxidizing acetyl CoA in the citric acid cycle? ✓ How is the citric acid cycle regulated? Y ou learned in Chapter 15 that glucose can be metabolized in glycolysis to pyruvate, yielding some ATP. However, the process of glycolysis is inefficient, capturing only a fraction of the energy inherent in a glucose molecule as ATP. More of the energy can be accessed if the pyruvate is completely oxidized to carbon dioxide and water. The combustion of fuels to carbon dioxide and water to generate ATP is called cellular respiration and is the source of more than 90% of the ATP required by human beings. Cellular respiration, unlike glycolysis, is an aerobic process, requiring molecular oxygen—O2. In eukaryotes, cellular respiration takes place inside the double-membrane bounded mitochondria, whereas glycolysis is cytoplasmic. Cellular respiration can be divided into two parts. First, carbon fuels are completely oxidized with a concomitant generation of high-transfer-potential electrons in a series of reactions variously called the citric acid cycle (CAC), the tricarboxylic acid (TCA) cycle, or the Krebs cycle. In the second part of cellular respiration, referred to as oxidative phosphorylation, the high-transfer-potential electrons are transferred to oxygen to form water in a series of oxidation–reduction reactions. This transfer is highly exergonic, and the released energy is used to synthesize ATP. We will focus on the citric acid cycle in this section, leaving oxidative phosphorylation until Section 9. The citric acid cycle is the central metabolic hub of the cell. It is the gateway to the aerobic metabolism of all fuel molecules. The cycle is also an important source of precursors for the building blocks of many other molecules such as amino acids, nucleotide bases, and porphyrin (the organic component of heme). The citric acid cycle component oxaloacetate also is an important precursor to glucose (p. 254). We begin this section by examining one of the most important reactions in living systems: the conversion of glucose-derived pyruvate into acetyl CoA, an activated acetyl unit and the actual substrate for the citric acid cycle. This reaction links glycolysis and cellular respiration, thus allowing for the complete combustion of glucose, a fundamental fuel in all living systems. We will then study the citric acid cycle itself, the final common pathway for the oxidation of all fuel molecules, carbohydrates, fats, and amino acids. Chapter 17: Preparation for the Cycle Chapter 18: Harvesting Electrons from the Cycle CHAPTER 17 Preparation for the Cycle 17.1 Pyruvate Dehydrogenase Forms Acetyl Coenzyme A from Pyruvate 17.2 The Pyruvate Dehydrogenase Complex Is Regulated by Two Mechanisms 17.3 The Disruption of Pyruvate Metabolism Is the Cause of Beriberi The majestic Brooklyn Bridge links Brooklyn with Manhattan in New York City. Pyruvate dehydrogenase links glycolysis with cellular respiration by converting pyruvate into acetyl CoA. Unlike the Brooklyn Bridge, however, molecular traffic flows in only one direction. [Ron Chapple Stock/Alamy.] A s you learned in Chapter 15, the pyruvate produced by glycolysis can have many fates. In the absence of oxygen (anaerobic conditions), the pyruvate is converted into lactic acid or ethanol, depending on the organism. In the presence of oxygen (aerobic conditions), it is converted into a molecule, called acetyl coenzyme A (acetyl CoA; Figure 17.1), that is able to enter the citric acid cycle. The path that pyruvate takes depends on the energy needs of the cell and the oxygen availability. In most tissues, pyruvate is processed aerobically because oxygen is readily available. For instance, in resting human muscle, most pyruvate is processed aerobically by first being converted into acetyl CoA. In very active muscle, however, much of the pyruvate is processed to lactate because the oxygen supply cannot meet the oxygen demand. 268 269 NH2 N O O – – O O P O O O 2 H O O O P – O OH O H N HN N O CH3 C N O P CH3 HO 17.1 Pyruvate Dehydrogenase N Figure 17.1 Coenzyme A. Coenzyme A is the activated carrier of acyl groups. Acetyl CoA, the fuel for the citric acid cycle, is formed by the pyruvate dehydrogenase complex. C S CH3 O Acetyl coenzyme A (Acetyl CoA) A schematic portrayal of the citric acid cycle is shown in Figure 17.2. The citric acid cycle accepts two-carbon acetyl units in the form of acetyl CoA. These two-carbon acetyl units are introduced into the cycle by binding to a four-carbon acceptor molecule. The two-carbon units are oxidized to CO2, and the resulting high-transfer-potential electrons are captured. The acceptor molecule is regenerated, capable of processing another two-carbon unit. The cyclic nature of these reactions enhances their efficiency. In this chapter, we examine the enzyme complex that catalyzes the formation of acetyl CoA from pyruvate, how this enzyme is regulated, and some pathologies that result if the function of the enzyme complex is impaired. 17.1 Pyruvate Dehydrogenase Forms Acetyl Coenzyme A from Pyruvate Glycolysis takes place in the cytoplasm of the cell, but the citric acid cycle takes place in the mitochondria (Figure 17.3). Pyruvate must therefore be transported into the mitochondria to be aerobically metabolized. This transport is facilitated by a special transporter (p. 324). In the mitochondrial matrix, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex to form acetyl CoA. Pyruvate + CoA + NAD + ¡ acetyl CoA + CO2 + NADH + H + Matrix Inner mitochondrial membrane Outer mitochondrial membrane Figure 17.3 Mitochondrion. The double membrane of the mitochondrion is evident in this electron micrograph. The oxidative decarboxylation of pyruvate and the sequence of reactions in the citric acid cycle take place within the matrix. [(Left) Omikron/Photo Researchers.] O H3C C Acetyl unit Four-carbon acceptor Six-carbon molecule GTP or ATP 2 CO2 High-transfer-potential electrons Figure 17.2 An overview of the citric acid cycle. The citric acid cycle oxidizes twocarbon units, producing two molecules of CO2, one molecule of GTP or ATP, and high-transfer-potential electrons. Table 17.1 Pyruvate dehydrogenase complex of E. coli Glucose Enzyme Abbreviation Number of chains Pyruvate dehydrogenase component E1 24 TPP Oxidative decarboxylation of pyruvate Dihydrolipoyl transacetylase E2 24 Lipoamide Transfer of acetyl group to CoA Dihydrolipoyl dehydrogenase E3 12 FAD Regeneration of the oxidized form of lipoamide Glycolysis Pyruvate CO2 Prosthetic group 2 e− Acetyl CoA Reaction cataiyzed Abbreviations: TPP, thiamine pyrophosphate; FAD, flavin adenine dinucleotide. Citric acid cycle 2 CO2 GTP This irreversible reaction is the link between glycolysis and the citric acid cycle (Figure 17.4). This reaction is a decisive reaction in metabolism: it commits the carbon atoms of carbohydrates to oxidation by the citric acid cycle or to the synthesis of lipids. Note that the pyruvate dehydrogenase complex produces CO 2 and captures high-transfer-potential electrons in the form of NADH, thus foreshadowing the key features of the reactions of the citric acid cycle. The pyruvate dehydrogenase complex is a large, highly integrated complex of three distinct enzymes (Table 17.1), each with its own active site. The pyruvate dehydrogenase complex is a member of a family of extremely large similar complexes with molecular masses ranging from 4 million to 10 million daltons (Figure 17.5). As we will see, their elaborate structures allow substrates to travel efficiently from one active site to another, connected by tethers to the core of the complex. 8 e− Figure 17.4 The link between glycolysis and the citric acid cycle. Pyruvate produced by glycolysis is converted into acetyl CoA, the fuel of the citric acid cycle. The Synthesis of Acetyl Coenzyme A from Pyruvate Requires Three Enzymes and Five Coenzymes We will examine the mechanism of action of the pyruvate dehydrogenase complex in some detail because it catalyzes a key juncture in metabolism—the link between glycolysis and the citric acid cycle that allows the complete oxidation of glucose. The mechanism of the pyruvate dehydrogenase reaction is wonderfully complex, more so than is suggested by its simple stoichiometry. The reaction requires the participation of the three enzymes of the pyruvate dehydrogenase complex as well as five coenzymes. The coenzymes thiamine pyrophosphate (TPP), lipoic acid, and FAD serve as catalytic coenzymes, and CoA and NAD ⫹ are stoichiometric coenzymes. Catalytic coenzymes, like enzymes, are not permanently altered by participation in the reaction. Stoichiometric coenzymes function as substrates. Figure 17.5 Electron micrograph of the pyruvate dehydrogenase complex from E. coli. [Courtesy of Dr. Lester Reed.] NH2 H + N N S S O H2N N O H3C O Thiamine pyrophosphate (TPP) 270 O P – P O O 2– S OH H O O Lipoic acid The conversion of pyruvate into acetyl CoA consists of three steps: decarboxylation, oxidation, and the transfer of the resultant acetyl group to CoA. O C H3C O CO2 C O C Decarboxylation – – H3C O 2 e– C Oxidation 17.1 Pyruvate Dehydrogenase O CoA + H3C 271 C H3C Transfer to CoA S CoA O Pyruvate Acetyl CoA These steps must be coupled to preserve the free energy derived from the decarboxylation step to drive the formation of NADH and acetyl CoA. 1. Decarboxylation. Pyruvate combines with the ionized (carbanion) form of TPP and is then decarboxylated to yield hydroxyethyl-TPP. R⬘ H3C N+ H R⬘ H3C N+ – + C + CO2 C CH3 S R TPP OH H O H3C Carbanion of TPP N+ Hydroxyethyl-TPP Pyruvate S R R⬘ H3C 2 H+ C O S R O – R⬘ H3C N+ – This reaction is catalyzed by the pyruvate dehydrogenase component (E1) of the multienzyme complex. TPP is the coenzyme of the pyruvate dehydrogenase component. S R Carbanion of TPP 2. Oxidation. The hydroxyethyl group attached to TPP is oxidized to form an acetyl group while being simultaneously transferred to lipoamide, a derivative of lipoic acid. Note that this transfer results in the formation of an energy-rich thioester bond. R⬘ H3C N+ OH – C S R – + CH3 S H Hydroxyethyl-TPP (ionized form) HS N+ S + H3C S R Lysine side chain S C H R⬙ O R⬙ Lipoamide O H N H R⬘ H3C + H+ Carbanion of TPP HN Acetyllipoamide O The disulfide group of lipoamide is reduced to its disulfhydryl form in this reaction. The reaction, also catalyzed by the pyruvate dehydrogenase component E1, yields acetyllipoamide. 3. Formation of Acetyl CoA. The acetyl group is transferred from acetyllipoamide to CoA to form acetyl CoA. Dihydrolipoyl transacetylase (E2) catalyzes this reaction. The energy-rich thioester bond is preserved as the acetyl group is transferred to CoA. Acetyl CoA, the fuel for the citric acid cycle, has now been generated from pyruvate. HS CoA SH + H3C C S C CoA H S R⬙ Acetyllipoamide CH3 + HS H O Coenzyme A HS O Acetyl CoA R⬙ Dihydrolipoamide H S S Reactive disulfide bond Lipoamide The pyruvate dehydrogenase complex must “reset” lipoamide so that the complex can catalyze another set of reactions. The complex cannot complete another catalytic cycle until the dihydrolipoamide is oxidized to lipoamide. In a fourth step, the oxidized form of lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3). Two electrons are transferred to an FAD prosthetic group of the enzyme and then to NAD⫹. 272 17 Preparation for the Cycle NAD+ HS S + FAD H H R⬙ Dihydrolipoamide E1(␣22) FAD + NADH + H+ S HS E3(␣) + FADH2 R⬙ Lipoamide This electron transfer from FAD to NAD⫹ is unusual, because the common role for FAD is to receive electrons from NADH. The electron-transfer potential of FAD is increased by its association with the enzyme, enabling it to transfer electrons to NAD ⫹ . Proteins tightly associated with FAD are called flavoproteins. Flexible Linkages Allow Lipoamide to Move Between Different Active Sites E2(␣3) Figure 17.6 A schematic representation of the pyruvate dehydrogenase complex. The transacetylase core (E2) is shown in red, the pyruvate dehydrogenase component (E1) in yellow, and the dihydrolipoyl dehydrogenase (E3) in green. The number and type of subunits of each enzyme is given parenthetically. The structures of all of the component enzymes of the pyruvate dehydrogenase complex are known, albeit from different complexes and species. Thus, it is now possible to construct an atomic model of the complex to understand its activity (Figure 17.6). The core of the complex is formed by the transacetylase component E2. Transacetylase consists of eight catalytic trimers assembled to form a hollow cube. Each of the three subunits forming a trimer has three major domains (Figure 17.7). At the amino terminus is a small domain that contains a flexible lipoamide cofactor. The lipoamide domain is followed by a small domain that Lipoamide domain Lipoamide Domain interacting with E3 component Figure 17.7 Structure of the transacetylase (E2) core. Each red ball represents a trimer of three E2 subunits. Notice that each subunit consists of three domains: a lipoamide-binding domain, a small domain for interaction with E3, and a large transacetylase catalytic domain. The transacetylase domain has three subunits, with one subunit depicted in red and the other two in white in the ribbon representation. A trimer Transacetylase domain NAD+ NADH + H+ FADH2 TPP Pyruvate CO2 FAD TPP H TPP 6 S 1 E1 S CH3 E3 S S E2 5 TPP S FAD TPP FAD OH C S 2 H FAD TPP SH HS S OH FAD C S CH3 O 3 4 S CH3 SH Acetyl CoA CoA Figure 17.8 Reactions of the pyruvate dehydrogenase complex. At the top (center), the enzyme (represented by a yellow, a green, and two red spheres) is unmodified and ready for a catalytic cycle. (1) Pyruvate is decarboxylated to form hydroxyethyl-TPP. (2) The lipoamide arm of E2 moves into the active site of E1. (3) E1 catalyzes the transfer of the two-carbon group to the lipoamide group to form the acetyl–lipoamide complex. (4) E2 catalyzes the transfer of the acetyl moiety to CoA to form the product acetyl CoA. The dihydrolipoamide arm then swings to the active site of E3. E3 catalyzes (5) the oxidation of the dihydrolipoamide acid and (6) the transfer of the protons and electrons to NAD⫹ to complete the reaction cycle. interacts with E3 within the complex. A larger transacetylase domain completes an E2 trimer. The eight E2 trimers are surrounded by twenty-four copies of E1 (an ␣22 tetramer ) and 12 copies of E3 (an ␣ dimer) surround the E2 core. How do the three distinct active sites work in concert? The key is the long, flexible lipoamide arm of the E2 subunit, which carries substrate from active site to active site (Figure 17.8). 1. Pyruvate is decarboxylated at the active site of E1, forming the hydroxyethylTPP intermediate, and CO2 leaves as the first product. This active site lies deep within the E1 complex, connected to the enzyme surface by a 20-Å-long hydrophobic channel. 2. E2 inserts the lipoamide arm of the lipoamide domain into the deep channel in E1 leading to the active site. 3. E1 catalyzes the transfer of the acetyl group to the lipoamide. The acetylated arm then leaves E1 and enters the E2 cube to visit the active site of E2, located deep in the cube at the subunit interface. 4. The acetyl moiety is then transferred to CoA, and the second product, acetyl CoA, leaves the cube. The reduced lipoamide arm then swings to the active site of the E3 flavoprotein. 273 274 17 Preparation for the Cycle 5. At the E3 active site, the lipoamide is oxidized by coenzyme FAD. The reactivated lipoamide is ready to begin another reaction cycle. 6. The final product, NADH, is produced with the reoxidation of FADH2 to FAD. The structural integration of three kinds of enzymes and the long flexible lipoamide arm make the coordinated catalysis of a complex reaction possible. The proximity of one enzyme to another increases the overall reaction rate and minimizes side reactions. All the intermediates in the oxidative decarboxylation of pyruvate remain bound to the complex throughout the reaction sequence and are readily transferred as the flexible arm of E 2 calls on each active site in turn. 17.2 The Pyruvate Dehydrogenase Complex Is Regulated by Two Mechanisms Glucose Pyruvate Pyruvate dehydrogenase complex Acetyl CoA CO2 Lipids Figure 17.9 From glucose to acetyl CoA. The synthesis of acetyl CoA by the pyruvate dehydrogenase complex is a key irreversible step in the metabolism of glucose. The pyruvate dehydrogenase complex is stringently regulated by multiple allosteric interactions and covalent modifications. As stated earlier, glucose can be formed from pyruvate through the gluconeogenic pathway (p. 251). However, the formation of acetyl CoA from pyruvate is an irreversible step in animals and thus they are unable to convert acetyl CoA back into glucose. The oxidative decarboxylation of pyruvate to acetyl CoA commits the carbon atoms of glucose to either of two principal fates: (1) oxidation to CO2 by the citric acid cycle with the concomitant generation of energy or (2) incorporation into lipid, inasmuch as acetyl CoA is a key precursor for lipid synthesis (Chapter 28 and Figure 17.9). High concentrations of reaction products inhibit the reaction: acetyl CoA inhibits the transacetylase component (E2) by directly binding to it, whereas NADH inhibits the dihydrolipoyl dehydrogenase (E3). High concentrations of NADH and acetyl CoA inform the enzyme that the energy needs of the cell have been met or that enough acetyl CoA and NADH have been produced from fatty acid degradation (p. 407). In either case, there is no need to metabolize pyruvate to acetyl CoA. This inhibition has the effect of sparing glucose, because most pyruvate is derived from glucose by glycolysis. The key means of regulation of the complex in eukaryotes is covalent modification—in this case, phosphorylation (Figure 17.10). Phosphorylation of the pyruvate dehydrogenase component (E1) by a specific kinase switches off the activity of the complex. Deactivation is reversed by the action of a specific phosphatase. Both the kinase and the phosphatase are physically associated with the transacetylase component (E2), again highlighting the structural and mechanistic importance of this core. Moreover, both the kinase and the phosphatase are themselves regulated. To see how this regulation works under biological conditions, consider muscle that is becoming active after a period of rest (Figure 17.11). At rest, the muscle will not have significant energy demands. Consequently, the NADH/NAD⫹, ATP ADP P Figure 17.10 The regulation of the pyruvate dehydrogenase complex. A specific kinase phosphorylates and inactivates pyruvate dehydrogenase (PDH), and a phosphatase activates the dehydrogenase by removing the phosphoryl group. The kinase and the phosphatase also are highly regulated enzymes. Kinase Active PDH Inactive PDH Phosphatase Pi H2O (A) HIGH ENERGY CHARGE Pyruvate (B) LOW ENERGY CHARGE NAD+ PDH + PDH − − − + NADH NADH Acetyl CoA CAC 17.3 Disruption of Pyruvate Metabolism Pyruvate NAD+ Acetyl CoA ADP e − ATP CAC 275 ADP ATP − e Figure 17.11 Response of the pyruvate dehydrogenase complex to the energy charge. The pyruvate dehydrogenase complex is regulated to respond to the energy charge of the cell. (A) The complex is inhibited by its immediate products, NADH and acetyl CoA, as well as by the ultimate product of cellular respiration, ATP. (B) The complex is activated by pyruvate and ADP, which inhibit the kinase that phosphorylates PDH. acetyl CoA/CoA, and ATP/ADP ratios will be high. These high ratios stimulate the kinase, promoting phosphorylation and, hence, deactivation of the pyruvate dehydrogenase complex. In other words, high concentrations of immediate (acetyl CoA and NADH) and ultimate (ATP) products of the pyruvate dehydrogenase complex inhibit its activity. Thus, pyruvate dehydrogenase is switched off when the energy charge is high. As exercise begins, the concentrations of ADP and pyruvate will increase as muscle contraction consumes ATP and glucose is converted into pyruvate to meet the energy demands. Both ADP and pyruvate activate the dehydrogenase by inhibiting the kinase. Moreover, the phosphatase is stimulated by Ca2⫹, a signal that also initiates muscle contraction. A rise in the cytoplasmic Ca2⫹ level to stimulate muscle contraction elevates the mitochondrial Ca2⫹ level. The rise in mitochondrial Ca2⫹ activates the phosphatase, enhancing pyruvate dehydrogenase activity. In some tissues, the phosphatase is regulated by hormones. In liver, epinephrine binds to the ␣-adrenergic receptor to initiate the phosphatidylinositol pathway (p. 180), causing an increase in Ca2⫹ concentration that activates the phosphatase. In tissues capable of fatty acid synthesis (such as the liver and adipose tissue), insulin (the hormone that signifies the fed state) stimulates the phosphatase, increasing the conversion of pyruvate into acetyl CoA. In these tissues, the pyruvate dehydrogenase complex is activated to funnel glucose to pyruvate and then to acetyl CoA and ultimately to fatty acids. Clinical Insight Defective Regulation of Pyruvate Dehydrogenase Results in a Pathological Condition In people with a phosphatase deficiency, pyruvate dehydrogenase is always phosphorylated and thus inactive. Consequently, glucose always has to take the anaerobic path and is processed to lactate rather than acetyl CoA. This condition results in unremitting lactic acidosis—high blood levels of lactic acid. In such an acidic environment, many tissues malfunction, most notably the central nervous system. ■ 17.3 The Disruption of Pyruvate Metabolism Is the Cause of Beriberi The importance of the coordinated activity of the pyruvate dehydrogenase complex is illustrated by disorders that result from the absence of a key coenzyme. Recall that thiamine pyrophosphate is a coenzyme for the pyruvate dehydrogenase activity of the pyruvate dehydrogenase complex. Beriberi, a neurological and cardiovascular disorder, is caused by a dietary deficiency of QUICK QUIZ List some of the advantages of organizing the enzymes that catalyze the formation of acetyl CoA from pyruvate into a single large complex. 276 17 Preparation for the Cycle “A certain very troublesome affliction, which attacks men, is called by the inhabitants Beriberi (which means sheep). I believe those, whom this same disease attacks, with their knees shaking and the legs raised up, walk like sheep. It is a kind of paralysis, or rather Tremor: for it penetrates the motion and sensation of the hands and feet indeed sometimes of the whole body.” —Jacob Bonitus, a physician working in Java in 1630 Figure 17.12 Milled and polished rice. Brown rice is milled to remove only the outer husk. Further milling (polishing) removes the inner husk also, resulting in white rice. [Image Source/Age Fotostock.] thiamine (also called vitamin B1). Thiamine deficiency results in insufficient pyruvate dehydrogenase activity because thiamine pyrophosphate cannot be formed. The disease has been and continues to be a serious health problem in the Far East because rice, the major food, has a rather low content of thiamine. This deficiency is partly ameliorated if the whole rice grain is soaked in water before milling; some of the thiamine in the husk then leaches into the rice kernel (Figure 17.12). The problem is exacerbated if the rice is polished, because only the outer layer contains significant amounts of thiamine. Beriberi is also occasionally seen in alcoholics who are severely malnourished and thus thiamine deficient. The disease is characterized by neurological and cardiac symptoms. Damage to the peripheral nervous system is expressed as pain in the limbs, weakness of the musculature, and distorted skin sensation. The heart may be enlarged and the cardiac output inadequate. Thiamine pyrophosphate is not just crucial to the conversion of pyruvate to acetyl CoA. In fact, this coenzyme is the prosthetic group of three important enzymes: pyruvate dehydrogenase, a-ketoglutarate dehydrogenase (a citric acid cycle enzyme, p. 283), and transketolase. Transketolase functions in the pentose phosphate pathway, which will be considered in Chapter 25. The common feature of enzymatic reactions utilizing TPP is the transfer of an activated aldehyde unit. As expected in a body in which TPP is deficient, the levels of pyruvate and ␣-ketoglutarate in the blood of patients with beriberi are higher than normal. The increase in the level of pyruvate in the blood is especially pronounced after the ingestion of glucose. A related finding is that the activities of the pyruvate dehydrogenase complex and the ␣-ketoglutarate dehydrogenase complex in vivo are abnormally low. The low transketolase activity of red blood cells in beriberi is an easily measured and reliable diagnostic indicator of the disease. Why does TPP deficiency lead primarily to neurological disorders? The nervous system relies essentially on glucose as its only fuel. The product of glycolysis— pyruvate—can enter the citric acid cycle only through the pyruvate dehydrogenase complex. With that enzyme deactivated, the nervous system has no source of fuel. In contrast, most other tissues can use fats as a source of fuel for the citric acid cycle. Symptoms similar to those of beriberi appear in organisms exposed to mercury or arsenite (AsO33-). Both substances have a high affinity for sulfhydryls in close proximity to one another, such as those in the reduced dihydrolipoyl groups of the E3 component of the pyruvate dehydrogenase complex (Figure 17.13). The binding of mercury or arsenite to the dihydrolipoyl groups inhibits the complex and leads to central nervous system pathologies. The proverbial phrase “mad 2,3-Dimercaptopropanol (BAL) HS S – O Excreted As HS S SH R H Dihydrolipoamide from pyruvate dehydrogenase component E3 As + Arsenite HO SH As O– HO HO S 2 H2O HO SH O – SH S R H Arsenite chelate on enzyme R H Restored enzyme Figure 17.13 Arsenite poisoning. Arsenite inhibits the pyruvate dehydrogenase complex by inactivating the dihydrolipoamide component of the transacetylase. Some sulfhydryl reagents, such as 2,3-dimercaptoethanol, relieve the inhibition by forming a complex with the arsenite that can be excreted. Figure 17.14 Mad Hatter. The Mad Hatter is one of the characters that Alice meets at a tea party in her journey through Wonderland. Real hatters worked with mercury, which inhibited an enzyme responsible for providing the brain with energy. The lack of energy would lead to peculiar behavior, often described as “mad.” [The Granger Collection.] as a hatter” refers to the strange behavior of poisoned hat makers who used mercury nitrate to soften and shape animal furs (Figure 17.14). This form of mercury is absorbed through the skin. Similar symptoms afflicted the early photographers, who used vaporized mercury to create daguerreotypes. Treatment for these poisons is the administration of sulfhydryl reagents with adjacent sulfhydryl groups to compete with the dihydrolipoyl residues for binding with the metal ion. The reagent–metal complex is then excreted in the urine. Indeed, 2,3-dimercaptopropanol (see Figure 17.13) was developed after World War I as an antidote to lewisite, an arsenic-based chemical weapon. This compound was initially called BAL, for British anti-lewisite. SUMMARY 17.1 Pyruvate Dehydrogenase Forms Acetyl Coenzyme A from Pyruvate Most fuel molecules enter the citric acid cycle as acetyl CoA. The link between glycolysis and the citric acid cycle is the oxidative decarboxylation of pyruvate to form acetyl CoA. In eukaryotes, this reaction and those of the cycle take place inside mitochondria, in contrast with glycolysis, which takes place in the cytoplasm. The enzyme complex catalyzing this reaction, the pyruvate dehydrogenase complex, consists of three distinct enzyme activities. Pyruvate dehydrogenase catalyzes the decarboxylation of pyruvate and the formation of acetyllipoamide. Dihydrolipoyl transacetylase forms acetyl CoA, and dihydrolipoyl dehydrogenase regenerates the active transacetylase. The complex requires five cofactors: thiamine pyrophosphate, lipoic acid, coenzyme A, NAD⫹, and FAD. 17.2 The Pyruvate Dehydrogenase Complex Is Regulated by Two Mechanisms The irreversible formation of acetyl CoA from pyruvate is an important regulatory point for the entry of glucose-derived pyruvate into the citric acid cycle. The pyruvate dehydrogenase complex is regulated by feedback inhibition by acetyl CoA and NADH. The activity of the pyruvate dehydrogenase complex is stringently controlled by reversible phosphorylation by an associated kinase and phosphatase. High concentrations of ATP and NADH stimulate the kinase, which phosphorylates and inactivates the complex. ADP and pyruvate inhibit the kinase, whereas Ca2⫹ stimulates the phosphatase, which dephosphorylates and thereby activates the complex. 17.3 The Disruption of Pyruvate Metabolism Is the Cause of Beriberi The importance of the pyruvate dehydrogenase complex to metabolism, especially to catabolism in the central nervous system, is illustrated by beriberi. Beriberi is a neurological condition that results from a deficiency of thiamine, the vitamin precursor of thiamine pyrophosphate. The lack of TPP impairs the activity of the pyruvate dehydrogenase component of the pyruvate dehydrogenase complex. Arsenite and mercury are toxic because of their effects on the complex. These chemicals bind to the lipoic acid coenzyme of dihydrolipoyl dehydrogenase, inhibiting the activity of this enzyme. 277 Summary 278 17 Preparation for the Cycle Key Terms lipoic acid (p. 270) pyruvate dehydrogenase (E1) (p. 271) acetyllipoamide (p. 271) dihydrolipoyl transacetylase (E2) (p. 271) acetyl CoA (p. 268) citric acid cycle (p. 268) pyruvate dehydrogenase complex (p. 269) thiamine pyrophosphate (TPP) (p. 270) dihydrolipoyl dehydrogenase (E3) (p. 272) flavoproteins (p. 272) beriberi (p. 275) Answer to QUICK QUIZ The advantages are as follows: 1. The reaction is facilitated by having the active sites in proximity. 2. The reactants do not leave the enzyme until the final product is formed. Constraining the reactants minimizes loss due to diffusion and minimizes side reactions. 3. All of the enzymes are present in the correct amounts. 4. Regulation is more efficient because the regulatory enzymes—the kinase and phosphatase—are part of the complex. Problems 1. Naming names. What are the five enzymes (including regulatory enzymes) that constitute the pyruvate dehydrogenase complex? Which reactions do they catalyze? 2. Coenzymes. What coenzymes are required by the pyruvate dehydrogenase complex and what are their roles? 3. More coenzymes. Distinguish between catalytic coenzymes and stoichiometric coenzymes in the pyruvate dehydrogenase complex. 4. A potent inhibitor. Thiamine thiazolone pyrophosphate binds to pyruvate dehydrogenase about 20,000 times as strongly as does thiamine pyrophosphate, and it competitively inhibits the enzyme. Why? R⬘ H3C H3C N+ R⬘ N H R S TPP O R S Thiazolone analog of TPP 5. Lactic acidosis. Patients in shock often suffer from lactic acidosis owing to a deficiency of O2. Why does a lack of O2 lead to lactic acid accumulation? One treatment for shock is to administer dichloroacetate, which inhibits the kinase associated with the pyruvate dehydrogenase complex. What is the biochemical rationale for this treatment? 6. Alternative fuels. As we will see (Chapter 26), fatty acid breakdown generates a large amount of acetyl CoA. What will be the effect of fatty acid breakdown on pyruvate dehydrogenase complex activity? On glycolysis? 7. Alternative fates. Compare the regulation of the pyruvate dehydrogenase complex in muscle and liver. 8. Mutations. (a) Predict the effect of a mutation that enhances the activity of the kinase associated with the PDH complex. (b) Predict the effect of a mutation that reduces the activity of the phosphatase associated with the PDH complex. 9. Flaking wallpaper. Claire Boothe Luce, Ambassador to Italy in the 1950s (and Connecticut congressperson, playwright, editor of Vanity Fair, and the wife of Henry Luce, founder of Time magazine and Sports Illustrated) became ill when she was staying at the ambassadorial residence in Italy. The wallpaper of her bedroom in the ambassadorial residence was colored a mellow green owing to the presence of cupric arsenite. Suggest a possible cause of Ambassador Luce’s illness. 10. Energy rich. What are the thioesters in the reaction catalyzed by PDH complex? Selected readings for this chapter can be found online at www.whfreeman.com/Tymoczko