CHAPTER 18 PREP. FOR THE CYCLE

18.1 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 mitochondria ( Figure 18.3 ). -Pyruvate must therefore be transported into mitochondria to be aerobically metabolized. -This transport is facilitated by a special transporter (Chapter 21). In the mitochondrial matrix, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex to form acetyl CoA.

Clinical Insight Enhanced Pyruvate Dehydrogenase Kinase Activity Facilitates the Development of Cancer

- Recall that cancer cells metabolize glucose to lactate even in the presence of oxy-gen, a phenomenon called aerobic glycolysis or the Warburg effect (p. 292). Un-der these conditions, the transcription factor hypoxia inducible factor-1 (HIF-1) increases the amount of the proteins required for glycolysis. - In addition, HIF-1 stimulates the production of pyruvate dehydrogenase kinase. The kinase inhib-its the pyruvate dehydrogenase complex, preventing the conversion of pyruvate into acetyl CoA. The pyruvate remains in the cytoplasm, further increasing the rate of aerobic glycolysis. - Moreover, even in the absence of increased synthesis of PDH kinase, mutations in PDH kinase have been identified that lead to enhanced activity, thereby contributing to increased aerobic glycolysis and the subsequent development of cancer as heretofore described. Enhanced lactate production re-sulting from aerobic glycolysis further enhances the activity of HIF-1.

Flexible Linkages Allow Lipoamide to Move Between Different Active Sites

- The structures of all of the component enzymes of the pyruvate dehydroge-nase 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.

-As you learned in Chapter 16, 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 18.1 ), 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 be-cause oxygen is readily available. - For instance, in resting human muscle, mostpyruvate is processed aerobically by first being converted into acetyl CoA. In very active muscle, however, much of the pyruvate is processed to lactate be-cause the oxygen supply cannot meet the oxygen demand.

-A schematic portrayal of the citric acid cycle is shown in Figure 18.2 . 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 CO 2 , and the resulting high-transfer-potential electrons are captured. The acceptor molecule is regener-ated, 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 complex is regulated, and some pathologies that result if the function of the enzyme complex is impaired. How-ever, the pyruvate dehydrogenase complex is not the only source of acetyl CoA. In particular, fatty acid degradation yields much acetyl CoA.

To see how this regulation works under biological conditions, consider mus-cle that is becoming active after a period of rest ( Figure 18.10 ). - At rest, the muscle will not have significant energy demands. Consequently, the NADH/NAD + , ace-tyl CoA/CoA, and ATP/ADP ratios will be high. These high ratios stimulate PDH kinase, promoting phosphorylation and, hence, deactivation of the pyruvate de-hydrogenase 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 PDH kinase. Moreover, the phosphatase is stimulated by Ca 2 + , a signal that also initiates muscle contraction. - A rise in the cytoplasmic Ca 2 + level to stimulate muscle contraction elevates the mitochondrial Ca 2 + level. -The rise in mitochondrial Ca 2 + activates the phosphatase, enhancing pyruvate dehydro-genase activity.

Clinical Insight Defective Regulation of Pyruvate Dehydrogenase Results in Lactic Acidosis

-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. One treatment for the condition is to place the patient on a ketogenic (high fat, adequate protein, low carbohydrate) diet to minimize the need to metabolize glucose. ■

The core of the complex is formed by the transacetylase component E2 . -Trans-acetylase consists of eight catalytic trimers assembled to form a hollow cube. Each of the three subunits forming a trimer has three major domains ( Figure 18.6 ). -At the amino terminus is a small domain that contains a flexible lipoamide cofactor covalently attached to a lysine side chain. -The lipoamide domain is followed by a small domain that interacts with E3 within the complex. A larger transacetylasedomain domain completes an E2 subunit.

-The eight E 2 trimers constitute the core of the complex and are surrounded by 24 copies of E 1 (an a 2 b 2 tetramer) and 12 copies of E 3 (an ab dimer). How do the three distinct active sites work in concert? The key is the long, flexible lipoamide arm of the E 2 subunit, which carries substrate from active site to active site ( Figure 18.7 ).

The conversion of pyruvate into acetyl CoA consists of three steps: decarboxylation, oxidation, and the transfer of the resultant acetyl group to 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.

-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 dehy-drogenase (E3). -Two electrons are transferred to an FAD prosthetic group of the enzyme and then to NAD + .

-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.

1. Decarboxylation Pyruvate combines with the ionized (carbanion) form of TPP and is then decarboxylated to yield hydroxyethyl-TPP.

-This reaction is catalyzed by the pyruvate dehydrogenase component (E 1) of the multienzyme complex. - TPP is the coenzyme of the pyruvate dehydrogenase component.

1. Pyruvate is decarboxylated at the active site of E 1 , forming the hydroxyethyl-TPP intermediate, and CO 2 leaves as the first product. This active site lies deep within the E 1 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 E 1 leading to the active site. 3. E 1 catalyzes the transfer of the acetyl group to the lipoamide. The acetylated arm then leaves E 1 and enters the E 2 cube to visit the active site of E 2 , 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 E 3 flavoprotein. 5. At the E 3 active site, the lipoamide is oxidized by coenzyme FAD. The reac-tivated lipoamide is ready to begin another reaction cycle. 6. The final product, NADH, is produced with the reoxidation of FADH 2 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 decarbox- ylation 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.

In some tissues, the phosphatase is regulated by hormones. In liver, epinephrine binds to the a -adrenergic receptor to initiate the phosphatidylino-sitol pathway (p. 222), causing an increase in Ca 2 + 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.

Figure 18.10 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.

-Recall that glycolysis generates two molecules of pyruvate for each glucose molecule metabolized. This irreversible conversion of pyruvate into acetyl CoA is the link between glycolysis and the citric acid cycle ( Figure 18.4 ). -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 (Chapter 29). 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.

Figure 18.4 -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. Fatty acid degradation is also an important source of acetyl CoA for the citric acid cycle (Chapter 27).

The key means of regulation of the complex in eukaryotes is covalent modification—in this case, phosphorylation ( Figure 18.9 ). -Phosphorylation of the pyruvate dehydrogenase component (E 1 ) by a pyruvate dehydrogenase (PDH) kinase switches off the activity of the complex. Deactivation is reversed by the ac-tion of a PDH phosphatase. -Both the kinase and the phosphatase are physically associated with the transacetylase component (E 2 ), again highlighting the struc-tural and mechanistic importance of this core. -Moreover, both the kinase and the phosphatase are themselves regulated.

Figure 18.8 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.

18.2 The Pyruvate Dehydrogenase Complex Is Regulated by Two Mechanisms -The pyruvate dehydrogenase complex is stringently regulated by multiple allo-steric interactions and covalent modifications. -As stated earlier, glucose can be formed from pyruvate through the gluconeogenic pathway (p. 302). -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 decar-boxylation of pyruvate to acetyl CoA commits the carbon atoms of glucose to either of two principal fates: ----(1) oxidation to CO 2 by the citric acid cycle with the concomitant generation of energy or ---(2) incorporation into lipid, because acetyl CoA is a key precursor for lipid synthesis (Chapter 28 and Figure 18.8 ).

High concentrations of reaction products inhibit the reaction: acetyl CoA inhibits the transacetylase component (E 2 ) by directly binding to it, whereas NADH inhibits the dihydrolipoyl dehydrogenase (E 3 ). -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 (Chapter 27). 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.

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.

The disulfide group of lipoamide is reduced to its disulfhydryl form in this reaction. The reaction, also catalyzed by the pyruvate dehydrogenase compo-nent E 1 , yields acetyllipoamide.

Clinical Insight 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 dehydroge-nase activity of the pyruvate dehydrogenase complex. Beriberi, a neurological and cardiovascular disorder, is caused by a dietary deficiency of thiamine (also called vitamin B 1 ). 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, an important food there, 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 18.11 ). - The problem is exacerbated if the rice is polished, a practice that prevents spoilage and extends the storage life of rice, because only the outer layer contains significant amounts of thiamine. Beriberi is also occasionally seen in alcoholics who are se-verely 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.

-Symptoms similar to those of beriberi appear in organisms exposed to mercury or arsenite (AsO3^ 3-) . -Both substances have a high affinity for sulfhydryls in close proximity to one another, such as those in the reduced dihydrolipoyl groups of the E 3 component of the pyruvate dehydrogenase complex ( Figure 18.12 ). -The binding of mercury or arsenite to the dihydrolipoyl groups inhibits the complex and leads to central nervous system pathologies. -The proverbial phrase "mad as a hatter" refers to the strange behavior of poisoned hat makers who used mercury nitrate to soften and shape animal furs ( Figure 18.13 ). -This form of mercury is ab-sorbed through the skin. Similar symptoms afflicted the early photographers, who used

Treatment for these poisons is the administration of sulfhydryl reagents with adjacent sulfhydryl groups to compete with the dihydrolipoyl residues for bind-ing with the metal ion. -The reagent-metal complex is then excreted in the urine. Indeed, 2,3-dimercaptopropanol (see Figure 18.12) was developed after World War I as an antidote to lewisite, an arsenic-based chemical weapon. This com-pound was initially called BAL, for British anti-lewisite.

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, α-ketoglutarate dehydrogenase (a citric acid cycle enzyme, Chapter 19), and transketolase. -Transketolase functions in the pentose phosphate pathway, which will be considered in Chapter 26. The common fea-ture 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 a -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 α-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.

Figure 18.3 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.]

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