Biological Chem | CH 17

17.1 The Pyruvate Dehydrogenase Complex Links Glycolysis to the Citric Acid Cycle

Carbohydrates, most notably glucose, are processed by glycolysis into pyruvate (Chapter 16). Under anaerobic conditions, the pyruvate is converted into lactate or ethanol, depending on the organism. Under aerobic conditions, the pyruvate is transported into mitochondria by a specific carrier protein embedded in the mitochondrial membrane. 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+

This irreversible reaction is the link between glycolysis and the citric acid cycle (Figure 17.4). Note that the pyruvate dehydrogenase complex produces CO2 and captures high-transfer-potential electrons in the form of NADH. Thus, the pyruvate dehydrogenase reaction has many of the key features of the reactions of the citric acid cycle itself.

The pyruvate dehydrogenase complex, a large, highly integrated complex of three distinct enzymes (Table 17.1), is another example of the organization of enzymes into supramolecular structures (p. 464). Pyruvate dehydrogenase complex is a member of a family of homologous complexes that include the citric acid cycle enzyme α- ketoglutarate dehydrogenase complex (p. 552). These complexes are giant, larger than ribosomes, with molecular masses ranging from 4 million to 10 million kDa. As we will see, their elaborate structures allow groups to travel from one active site to another, connected by tethers to the core of the structure.

TABLE 17.1 Pyruvate dehydrogenase complex of E. coli

Enzyme Abbreviation Prosthetic Reaction group catalyzed

Pyruvate E1 TPP dehydrogenase
component

Oxidative decarboxylation of pyruvate

Dihydrolipoyl E2 Lipoamide Transfer of transacetylase acetyl group to

CoA

Dihydrolipoyl E3 FAD dehydrogenase

Regeneration of the oxidized form of lipoamide

Mechanism: The synthesis of acetyl coenzyme A from pyruvate requires three enzymes and five coenzymes

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 and five coenzymes. The coenzymes thiamine pyrophosphate (TPP), lipoic acid, and FAD serve as catalytic cofactors, and CoA and NAD+ are stoichiometric cofactors, cofactors that function as substrates.

The conversion of pyruvate into acetyl CoA consists of three steps: decarboxylation, oxidation, and transfer of the resultant acetyl group to CoA. A fourth step is required to regenerate the active enzyme.

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.PyruvatecombineswithTPPandisthen decarboxylated to yield hydroxyethyl-TPP (Figure 17.5).

FIGURE 17.5 Mechanism of the E1 decarboxylation reaction. E1 is the pyruvate dehydrogenase component of the pyruvate dehydrogenase complex. A key feature of the prosthetic group, TPP, is that the carbon atom between the nitrogen and sulfur atoms in the

thiazole ring is much more acidic than most ═ C— groups, with a pKa value near 10. (1) This carbon center ionizes to form a carbanion. (2) The carbanion readily adds to the carbonyl group of pyruvate. (3) This addition is followed by the decarboxylation of pyruvate. The positively charged ring of TPP acts as an electron sink that stabilizes the negative charge that is transferred to the ring as part of the decarboxylation. (4) Protonation yields hydroxyethyl-TPP.

This reaction, the rate-limiting step in acetyl CoA synthesis, is catalyzed by the pyruvate dehydrogenase component (E1) of the

multienzyme complex. TPP is the prosthetic group of the pyruvate dehydrogenase component.

2. Oxidation.ThehydroxyethylgroupattachedtoTPPisoxidizedto form an acetyl group while being simultaneously transferred to lipoamide, a derivative of lipoic acid that is linked to the side chain of a lysine residue by an amide linkage. Note that this transfer results in the formation of an energy-rich thioester bond.

The oxidant in this reaction is the disulfide group of lipoamide, which is reduced to its disulfhydryl form. This reaction, also catalyzed by the pyruvate dehydrogenase component E1, yields acetyllipoamide.

3. FormationofAcetylCoA.Theacetylgroupistransferredfrom 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. Recall that CoA serves as a carrier of many activated acyl groups, of which acetyl is the simplest (Section 15.3).

Acetyl CoA, the fuel for the citric acid cycle, has now been generated from pyruvate.

4. Regenerationofoxidizedlipoamide.Thepyruvatedehydrogenase complex cannot complete another catalytic cycle until the dihydrolipoamide is oxidized to lipoamide. In the 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+.

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 chemical environment within the enzyme, enabling it to transfer electrons to NAD+. Proteins tightly associated with FAD or flavin mononucleotide (FMN) are called flavoproteins.

Flexible linkages allow lipoamide to move between different active sites

The structures and precise composition of the component enzymes of the pyruvate dehydrogenase vary among species. However, there are commonalities.

The core of the complex is formed by 60 molecules of the transacetylase component E2 (Figure 17.6). Transacetylase consists of

20 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 bound flexible lipoamide cofactor attached to a lysine residue. This domain is homologous to biotin-binding domains such as that of pyruvate carboxylase (Figure 16.25). The lipoamide domain is followed by a small domain that interacts with E3 within the complex. A larger transacetylase domain completes an E2 subunit. Surrounding the core transacetylase is a shell composed of ∼45 copies of the E1 and ∼10 copies of E3 enzymes. In mammals, E1 is an α2β2 tetramer, and E3 is an αβ dimer, and this core contains another protein, E3-

which facilitates the interaction between E2 and E3. If E3-BP is missing, the

complex has greatly reduced activity. The gap between the outer shell and the transacetylase core allows the lipoamide arms to visit the various active sites as described below (Figure 17.6).

binding protein (E3-BP),

FIGURE 17.6 Structure of the pyruvate dehydrogenase complex from B. stearothermophilus. The image of the complex, which was derived from cryo-electron microscopic data (Section 3.5), shows an inner core consisting

of the E2 enzyme. The shell surrounding the

core consists of E1 and E3 enzymes,

although only the E1 enzymes are shown in this structure. Two of the 60 lipoamide arms are shown (red and yellow).

interact with one another to form the catalytic trimer. Transacetylase domains of three identical subunits are shown, with one depicted in red and the others in white in the ribbon representation.

How do the three distinct active sites work in concert (Figure 17.8)? The key is the long, flexible lipoamide arm of the E2 subunit, which carries substrate from active site to active site:

1. PyruvateisdecarboxylatedattheactivesiteofE1, formingthe hydroxyethyl-TPP 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. TheacetylmoietyisthentransferredtoCoA,andthesecond product, acetyl CoA, leaves the cube. The reduced lipoamide arm then swings to the active site of the E3 flavoprotein.
5. At the E3 active site, the lipoamide is oxidized by coenzyme FAD. The reactivated lipoamide is ready to begin another reaction cycle.
6. Thefinalproduct,NADH,isproducedwiththereoxidationof 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 E2 calls on each active site in turn.

17.2 The Citric Acid Cycle Oxidizes Two-Carbon Units

The conversion of pyruvate into acetyl CoA by the pyruvate dehydrogenase complex is the link between glycolysis and cellular respiration because acetyl CoA is the fuel for the citric acid cycle. Indeed, all fuels are ultimately metabolized to acetyl CoA or components of the citric acid cycle.

Citrate synthase forms citrate from oxaloacetate and acetyl coenzyme A

The citric acid cycle begins with the condensation of a four-carbon unit, oxaloacetate, and a two-carbon unit, the acetyl group of acetyl CoA. Oxaloacetate reacts with acetyl CoA and H2O to yield citrate and CoA.

This reaction, which is an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, a molecule that is energy rich because it

contains the thioester bond that originated in acetyl CoA. The hydrolysis of citryl CoA thioester to citrate and CoA drives the overall reaction far in the direction of the synthesis of citrate. In essence, the hydrolysis of the thioester powers the synthesis of a new molecule from two precursors.

Mechanism: The mechanism of citrate synthase prevents undesirable reactions

Because the condensation of acetyl CoA and oxaloacetate initiates the citric acid cycle, it is very important that side reactions, notably the hydrolysis of acetyl CoA to acetate and CoA, be minimized. Let us briefly consider how the citrate synthase prevents the wasteful hydrolysis of acetyl CoA.

Synthase

An enzyme catalyzing a synthetic reaction in which two units are joined usually without the direct participation of ATP (or another nucleoside triphosphate).

Mammalian citrate synthase is a dimer of identical 49-kDa subunits. Each active site is located in a cleft between the large and the small domains of a subunit, adjacent to the subunit interface. X-ray

crystallographic studies of citrate synthase and its complexes with several substrates and inhibitors have revealed that the enzyme undergoes large conformational changes in the course of catalysis. Citrate synthase exhibits sequential, ordered kinetics: oxaloacetate binds first, followed by acetyl CoA. The reason for the ordered binding is that oxaloacetate induces a major structural rearrangement leading to the creation of a binding site for acetyl CoA. The binding of oxaloacetate converts the open form of the enzyme into a more closed form (Figure 17.9). In each subunit, the small domain rotates 19 degrees relative to the large domain. Movements as large as 15 Å are produced by the rotation of α helices elicited by quite small shifts of side chains around bound oxaloacetate. These structural changes create a binding site for acetyl CoA.

Citrate synthase catalyzes the condensation reaction by bringing the substrates into close proximity, orienting them, and polarizing certain bonds (Figure 17.10). The donation and removal of protons transforms acetyl CoA into an enol intermediate. The enol attacks oxaloacetate to form a carbon–carbon double bond linking acetyl CoA and oxaloacetate. The newly formed citryl CoA induces additional structural changes in the enzyme, causing the active site to become completely enclosed. The enzyme cleaves the citryl CoA thioester by hydrolysis. CoA leaves the enzyme, followed by citrate, and the enzyme returns to the initial open conformation.

We can now understand how the wasteful hydrolysis of acetyl CoA is prevented. Citrate synthase is well suited to hydrolyze citryl CoA but not acetyl CoA. How is this discrimination accomplished? First, acetyl CoA does not bind to the enzyme until oxaloacetate is bound and ready for condensation. Second, the catalytic residues crucial for the hydrolysis of the thioester linkage are not appropriately positioned until citryl CoA is formed. As with hexokinase and triose phosphate isomerase (Section 16.1), induced fit prevents an undesirable side reaction.

Citrate is isomerized into isocitrate

The hydroxyl group is not properly located in the citrate molecule for the oxidative decarboxylations that follow (problem 31). Thus, citrate is isomerized into isocitrate to enable the six-carbon unit to undergo oxidative decarboxylation. The isomerization of citrate is accomplished by a dehydration step followed by a hydration step. The result is an interchange of an H and an OH. The enzyme catalyzing both steps is called aconitase because cis-aconitate is an intermediate.

Aconitase is an iron–sulfur protein, or nonheme-iron protein, in that it contains iron that is not bonded to heme. Rather, its four iron atoms are

complexed to four inorganic sulfides and three cysteine sulfur atoms, leaving one iron atom available to bind citrate through one of its COO- groups and an OH group (Figure 17.11). This Fe-S cluster

participates in dehydrating and rehydrating the bound substrate.

Isocitrate is oxidized and decarboxylated to alpha- ketoglutarate

We come now to the first of four oxidation–reduction reactions in the citric acid cycle. The oxidative decarboxylation of isocitrate is catalyzed by isocitrate dehydrogenase.

Isocitrate + NAD+→α-ketoglutarate+CO2+NADH

The intermediate in this reaction is oxalosuccinate, an unstable β- ketoacid. While bound to the enzyme, it loses CO2 to form α-ketoglutarate.

This oxidation generates the first high-transfer-potential electron carrier, NADH, in the cycle.

Succinyl coenzyme A is formed by the oxidative decarboxylation of alpha- ketoglutarate

The conversion of isocitrate into α-ketoglutarate is followed by a second oxidative decarboxylation reaction, the formation of succinyl CoA from α-ketoglutarate.

This reaction is catalyzed by the α-ketoglutarate dehydrogenase complex, an organized assembly of three kinds of enzymes that is

homologous to the pyruvate dehydrogenase complex. In fact, the E3 component is identical in both enzymes. The oxidative decarboxylation of α-ketoglutarate closely resembles that of pyruvate, also an α-ketoacid.

Pyruvate + CoA + NAD+⟶Pyruvate dehydrogenase complexacetyl CoA + CO2

α-Ketoglutarate + CoA + NAD+⟶α-
Ketoglutarate dehydrogenase complexsuccinyl CoA + CO2+NADH

Both reactions include the decarboxylation of an α-ketoacid and the subsequent formation of a thioester linkage with CoA that has a high transfer potential. The reaction mechanisms are entirely analogous (p. 544).

A compound with high phosphoryl-

transfer potential is generated from succinyl coenzyme A

Succinyl CoA is an energy-rich thioester compound. The ΔG°′ for the hydrolysis of succinyl CoA is about -33.5 kJ mol-1 (-8.0 kcal mol-1),

which is comparable to that of ATP

(-30.5 kJ mol-1, or -7.3 kcal mol-1). In the citrate synthase reaction, the cleavage of the thioester bond powers the synthesis of the six-carbon citrate from the four-carbon oxaloacetate and the two-carbon fragment. The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate, usually ADP. This reaction, which is readily reversible, is catalyzed by succinyl CoA synthetase (also called, succinate thiokinase).

This reaction is the only step in the citric acid cycle that directly yields a compound with high phosphoryl-transfer potential. In mammals, there are two isozymic forms of the enzyme, one specific for ADP and one for GDP. In tissues that perform large amounts of cellular respiration, such as skeletal and heart muscle, the ADP-requiring isozyme predominates. In tissues that perform many anabolic reactions, such as the liver, the GDP-requiring enzyme is common. The GDP-

requiring enzyme is believed to work in reverse of the direction observed in the TCA cycle; that is, GTP is used to power the synthesis of succinyl CoA, which is a precursor for heme synthesis (Section 24.4). The E. coli enzyme uses either GDP or ADP as the phosphoryl-group acceptor.

Note that the enzyme nucleoside diphosphokinase, which catalyzes the following reaction,

GTP + ADP⇌GDP + ATP

allows the γ phosphoryl group to be readily transferred from GTP to form ATP, thereby allowing the adjustment of the concentration of GTP or ATP to meet the cell’s need.

Mechanism: Succinyl coenzyme A synthetase transforms types of biochemical energy

The mechanism of this reaction is a clear example of an energy transformation: energy inherent in the thioester molecule is transformed into phosphoryl-group-transfer potential (Figure 17.12). The first step is the displacement of coenzyme A by orthophosphate, which generates another energy-rich compound, succinyl phosphate. A histidine residue plays a key role as a moving arm that detaches the phosphoryl group, then swings over to a bound ADP and transfers the

group to form ATP. The participation of high-energy compounds in all the steps is attested to by the fact that the reaction is readily reversible:

ΔG°′=-3.4 kJ mol-1 (-0.8 kcal mol-1).

The formation of ATP at the expense of succinyl CoA is an example of substrate-level phosphorylation (Section 16.1).

Oxaloacetate is regenerated by the oxidation of succinate

Reactions of four-carbon compounds constitute the final stage of the citric acid cycle: the regeneration of oxaloacetate.

The reactions constitute a metabolic motif that we will see again in fatty acid synthesis and degradation as well as in the degradation of some amino acids. A methylene group (CH2) is converted into a carbonyl

group C═O in three steps: an oxidation, a hydration, and a second oxidation reaction. Oxaloacetate is thereby regenerated for another round of the cycle, and more energy is extracted in the form of FADH2

and NADH.

Succinate is oxidized to fumarate by succinate dehydrogenase. The hydrogen acceptor is FAD rather than NAD+, which is used in the

other three oxidation reactions in the cycle. FAD is the hydrogen acceptor in this reaction because the free-energy change is insufficient to reduce NAD+. FAD is nearly always the electron acceptor in oxidations that remove two hydrogen atoms from a substrate. In succinate dehydrogenase, the isoalloxazine ring of FAD is covalently attached to a histidine side chain of the enzyme (denoted E-FAD).

E-FAD + succinate⇌E-FADH2 + fumarate

Succinate dehydrogenase, like aconitase, is an iron–sulfur protein. Indeed, succinate dehydrogenase contains three different kinds of iron– sulfur clusters: 2Fe-2S (two iron atoms bonded to two inorganic sulfides), 3Fe-4S, and 4Fe-4S. Succinate dehydrogenase—which consists

of a 70-kDa and a 27-kDa subunit—differs from other enzymes in the citric acid cycle because it is embedded in the inner mitochondrial membrane. In fact, succinate dehydrogenase is directly associated with the electron-transport chain, the link between the citric acid cycle and ATP formation. FADH2 produced by the oxidation of succinate does not dissociate from the enzyme, in contrast with NADH produced in other oxidation–reduction reactions. Rather, two electrons are transferred from FADH2 directly to iron–sulfur clusters of the enzyme, which in turn passes the electrons to coenzyme Q (CoQ). Coenzyme Q, an important member of the electron-transport chain, passes electrons to the ultimate acceptor, molecular oxygen, as we shall see in Chapter 18.

The next step is the hydration of fumarate to form L-malate. Fumarase catalyzes a stereospecific trans addition of H+ and OH-. The OH- group adds to only one side of the double bond of

fumarate; hence, only the L isomer of malate is formed.

Finally, malate is oxidized to form oxaloacetate. This reaction is catalyzed by malate dehydrogenase, and NAD+ is again the hydrogen acceptor.

Malate + NAD+⇌oxaloacetate+NADH+H+

The standard free energy for this reaction, unlike that for the other steps in the citric acid cycle, is significantly positive ( ΔG°′ =+29.7 kJ mol-

1, or +7.1 kcal mol-1). The oxidation of malate is driven by the use of the products—oxaloacetate by citrate synthase and NADH by the electron-transport chain.

The citric acid cycle produces high- transfer-potential electrons, ATP, and CO2

The net reaction of the citric acid cycle is

Acetyl CoA + 3 NAD+ + FAD + ADP + Pi + 2 H2O→2 CO2 + 3 NADH + FADH

Let us recapitulate the reactions that give this stoichiometry (Figure 17.13 and Table 17.2):

1. 
   Twocarbonatomsenterthecycleinthecondensationofanacetyl unit (from acetyl CoA) with oxaloacetate. Two carbon atoms leave the cycle in the form of CO2 in the successive decarboxylations catalyzed by isocitrate dehydrogenase and α-ketoglutarate
   dehydrogenase.
2. Fourpairsofhydrogenatomsleavethecycleinfouroxidation reactions. Two NAD+ molecules are reduced in the oxidative
   decarboxylations of isocitrate and α-ketoglutarate, one FAD molecule is reduced in the oxidation of succinate, and one NAD+ molecule is reduced in the oxidation of malate. Recall also that one NAD+ molecule is reduced in the oxidative decarboxylation of pyruvate to form acetyl CoA.

1. Onecompoundwithhighphosphoryl-transferpotential,usually ATP, is generated from the cleavage of the thioester linkage in succinyl CoA.
2. Twowatermoleculesareconsumed:oneinthesynthesisofcitrate by the hydrolysis of citryl CoA and the other in the hydration of fumarate.

Isotope-labeling studies have revealed that the two carbon atoms that enter each cycle are not the ones that leave. The two carbon atoms that enter the cycle as the acetyl group are retained during the initial two decarboxylation reactions (Figure 17.13) and then remain incorporated in the four-carbon acids of the cycle. Note that succinate is a symmetric molecule. Consequently, the two carbon atoms that enter the cycle can occupy any of the carbon positions in the subsequent metabolism of the four-carbon acids. The two carbons that enter the cycle as the acetyl group will be released as CO2 in subsequent trips through the cycle. To understand why citrate is not processed as a symmetric molecule, see problems 38 and 39.

Various techniques, such as fluorescence recovery after photobleaching (FRAP, Section 12.5) and tandem mass spectroscopy analysis (Section 3.3), have established that there is a physical association of all of the enzymes of the citric acid cycle into a supramolecular complex. The close arrangement of enzymes enhances the efficiency of the citric acid cycle because a reaction product can pass directly from one active site to the next through connecting channels, a process called substrate channeling.

As will be considered in Chapter 18, the electron-transport chain oxidizes the NADH and FADH2 formed in the citric acid cycle.

The transfer of electrons from these carriers to O2, the ultimate electron acceptor, leads to the generation of a proton gradient across the inner mitochondrial membrane. This proton-motive force then powers the generation of ATP; the net stoichiometry is about 2.5 ATP per NADH, and 1.5 ATP per FADH2. Consequently, nine high-transfer- potential phosphoryl groups are generated when the electron-transport chain oxidizes 3 NADH molecules and 1 FADH2 molecule, and one ATP is directly formed in one round of the citric acid cycle. Thus, one acetyl unit generates approximately 10 molecules of ATP. In dramatic contrast, the anaerobic glycolysis of an entire glucose molecule generates only 2 molecules of ATP (and 2 molecules of lactate).

Recall that molecular oxygen does not participate directly in the citric acid cycle. However, the cycle operates only under aerobic conditions because NAD+ and FAD can be regenerated in the mitochondrion only by the transfer of electrons to molecular oxygen. Glycolysis has both an aerobic and an anaerobic mode, whereas the citric acid cycle is strictly aerobic. Glycolysis can proceed under anaerobic conditions because NAD+ is regenerated in the conversion of pyruvate into lactate or ethanol.

17.3 Entry to the Citric Acid Cycle and Metabolism Through It Are Controlled

The citric acid cycle is the final common pathway for the aerobic oxidation of fuel molecules. Moreover, as we will see shortly (Section 17.4) and repeatedly elsewhere in our study of biochemistry, the cycle is an important source of building blocks for a host of important biomolecules. As befits its role as the metabolic hub of the cell, entry into the cycle and the rate of the cycle itself are controlled at several stages.

The pyruvate dehydrogenase complex is regulated allosterically and by reversible phosphorylation

As stated earlier, glucose can be formed from pyruvate (Section 16.3). 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 one of two principal fates: oxidation to CO2 by the citric acid cycle, with the concomitant generation of energy, or incorporation into lipid (Figure 17.14). As expected of an enzyme at a critical branch point in metabolism, the activity of the pyruvate dehydrogenase complex is stringently controlled. High concentrations of reaction products inhibit the

reaction: acetyl CoA inhibits the transacetylase component (E2) by binding directly, 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 fatty acids are being degraded to produce acetyl CoA and NADH. 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 (Section 16.1).

EXPLORE this pathway further in the Metabolic Map on

The key means of regulation of the complex in eukaryotes is covalent modification (Figure 17.15). Phosphorylation of the pyruvate dehydrogenase component (E1) by pyruvate dehydrogenase kinase (PDK) switches off the activity of the complex. There are four isozymes of PDK that are expressed in a tissue-specific manner. Deactivation is reversed by the pyruvate dehydrogenase phosphatase (PDP), of which there are two isozymic forms. In mammals, the kinase and the phosphatase are associated with the E2-E3-BP complex, again highlighting the structural and mechanistic importance of this core. Both the kinase and the phosphatase are regulated. To see how this regulation works in biological conditions, consider muscle that is becoming active after a period of rest (Figure 17.16). At rest, the muscle will not have significant energy demands. Consequently, the NADH/NAD+, acetyl CoA/CoA, and ATP/ADP ratios will be high. These high ratios promote phosphorylation and inactivation of the complex by activating PDK. In other words, high concentrations of immediate (acetyl CoA and NADH) and ultimate (ATP) products inhibit the activity. Thus, pyruvate dehydrogenase is switched off when the energy charge is high.

inhibit the kinase that phosphorylates PDH.

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+, the same signal that initiates muscle contraction. A rise in the cytoplasmic Ca2+ level (See Section 36.2 on ) 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 α-adrenergic receptor to initiate the phosphatidylinositol pathway (Section 14.1), 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. Acetyl CoA is the precursor for fatty acid synthesis (Section 22.4). In these tissues, the pyruvate dehydrogenase complex is activated to funnel glucose to pyruvate and then to acetyl CoA and ultimately to fatty acids.

In people with a pyruvate dehydrogenase phosphatase deficiency,

pyruvate dehydrogenase is always phosphorylated and thus inactive. Consequently, glucose 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 (problem 18). See Biochemistry in Focus (p. 566) for a discussion of the role of the role of pyruvate dehydrogenase complex in diabetic neuropathy.

The citric acid cycle is controlled at several points

The rate of the citric acid cycle is precisely adjusted to meet an animal cell’s needs for ATP (Figure 17.17). The primary control points are the allosteric enzymes isocitrate dehydrogenase and α-ketoglutarate

dehydrogenase, the first two enzymes in the cycle to generate high-energy electrons.

FIGURE 17.17 Control of the citric acid cycle. The citric acid cycle is regulated primarily by the concentration of ATP and NADH. The key control points are the enzymes isocitrate dehydrogenase

and α-ketoglutarate dehydrogenase.

The first control site is isocitrate dehydrogenase. The enzyme is allosterically stimulated by ADP, which enhances the enzyme’s affinity

for substrates. The binding of isocitrate, NAD+, Mg2+, and ADP is mutually cooperative. In contrast, ATP is inhibitory. The reaction product NADH also inhibits isocitrate dehydrogenase by directly displacing NAD+. It is important to note that several steps in the cycle require NAD+ or FAD, which are abundant only when the energy charge is low.

A second control site in the citric acid cycle is α-ketoglutarate

dehydrogenase, which catalyzes the rate-limiting step in the citric acid cycle. Some aspects of this enzyme’s control are like those of the pyruvate dehydrogenase complex, as might be expected from the homology of the two enzymes. α-Ketoglutarate
dehydrogenase is inhibited by succinyl CoA and NADH, the products of the reaction that it catalyzes. In addition, α-Ketoglutarate
dehydrogenase is inhibited by a high energy charge. Thus, the rate of the cycle is reduced when the cell has a high level of ATP. α-Ketoglutarate

dehydrogenase deficiency is observed in a number of neurological disorders, including Alzheimer’s disease.

The use of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase as control points integrates the citric acid cycle with other pathways and highlights the central role of the citric acid cycle in metabolism. For instance, the inhibition of isocitrate dehydrogenase leads to a buildup of citrate, because the interconversion of isocitrate and citrate is readily reversible under intracellular conditions. Citrate can be transported to the cytoplasm, where it signals phosphofructokinase to halt glycolysis (Section 16.2) and where it can serve as a source of acetyl CoA for fatty acid synthesis (Section 22.4). The α-ketoglutarate that accumulates when α-ketoglutarate

dehydrogenase is inhibited can be used as a precursor for several amino acids and the purine bases (Chapter 23 and Chapter 25).

In many bacteria, the funneling of two-carbon fragments into the cycle also is controlled. The synthesis of citrate from oxaloacetate and acetyl CoA carbon units is an important control point in these organisms. ATP is an allosteric inhibitor of citrate synthase. The effect of ATP is to increase the value of KM for acetyl CoA. Thus, as the level of ATP increases, less of this enzyme is bound to acetyl CoA and so less citrate is formed.

Defects in the citric acid cycle contribute to the development of cancer

Four enzymes crucial to cellular respiration are known to

contribute to the development of cancer: succinate dehydrogenase, fumarase, pyruvate dehydrogenase kinase, and isocitrate dehydrogenase. Mutations that alter the activity of the first three of these enzymes enhance aerobic glycolysis (Section 16.2). In aerobic glycolysis, cancer cells preferentially metabolize glucose to lactate even in the presence of oxygen. Defects in these enzymes share a common biochemical link: the transcription factor hypoxia inducible factor 1 (HIF-1).

Normally, HIF-1 up-regulates the enzymes and transporters that enhance glycolysis only when oxygen concentration falls, a condition called hypoxia. Under normal conditions, HIF-1 is hydroxylated by prolyl hydroxylase 2 and is subsequently destroyed by the proteasome, a large complex of proteolytic enzymes (Section 23.2). The degradation of HIF-1 prevents the stimulation of glycolysis. Prolyl hydroxylase 2 requires α-ketoglutarate, ascorbate (Vitamin C), and oxygen for activity. Thus, when oxygen concentration falls, the prolyl hydroxylase 2 is inactive, HIF-1 is not hydroxylated and not degraded, and the synthesis of proteins required for glycolysis is stimulated. As a result, the rate of glycolysis is increased.

Recent research suggestions that defects in the enzymes of the citric acid cycle may significantly affect the regulation of prolyl hydroxylase 2. When either succinate dehydrogenase or fumarase is defective, succinate and fumarate accumulate in the mitochondria and spill over into the cytoplasm. Both succinate and fumarate are competitive inhibitors of prolyl hydroxylase 2. The inhibition of prolyl hydroxylase 2

results in the stabilization of HIF-1, since HIF-1 is no longer hydroxylated. Lactate, the end product of glycolysis, also appears to inhibit prolyl hydroxylase 2 by interfering with the action of ascorbate. In addition to increasing the amount of the proteins required for glycolysis, HIF-1 also stimulates the production of pyruvate dehydrogenase kinase (PDK). The kinase inhibits 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, mutations in PDK that lead to enhanced activity contribute to increased aerobic glycolysis and the subsequent development of cancer. By enhancing glycolysis and increasing the concentration of lactate, the mutations in PDK result in the inhibition of hydroxylase and the stabilization of HIF-1.

Mutations in isocitrate dehydrogenase result in the generation of an oncogenic metabolite, 2-hydroxyglutarate. The mutant enzyme catalyzes the conversion of isocitrate to α-ketoglutarate, but then reduces α-ketoglutarate to form 2-hydroxyglutarate. 2- Hydroxyglutarate alters the methylation patterns in DNA (Section 33.3) and reduces dependence on growth factors for proliferation. These changes alter gene expression and promote unrestrained cell growth.

An enzyme in lipid metabolism is hijacked to inhibit pyruvate dehydrogenase activity

A particularly fascinating example of the manipulation of cellular

control by cancer cells comes from recent findings on the mitochondrial enzyme acetyl CoA acetyltransferase. Under normal circumstances, this enzyme synthesizes ketone bodies, such as acetoacetate, which are a fuel source for some tissues and an essential fuel source for all tissues under starvation conditions (Section 22.3)

2 Acetyl CoA+H2O⟶ Acetyl CoAacetyltransferaceacetoacetate+2 CoA

In certain cancers, acetyl CoA acetyltransferase is phosphorylated, which causes the enzyme to form active tetramers. Interestingly, the enzyme then acts as a protein acetyltransferase, adding acetyl groups to pyruvate dehydrogenase and pyruvate dehydrogenase phosphatase. The acetylation inhibits the two enzymes and facilitates the metabolic switch from oxidative phosphorylation to aerobic glycolysis, thereby enhancing the Warburg effect (Section 16.2). In essence, the enzyme is hijacked from its normal role in ketone body formation to further cancer growth, a remarkable occurrence. The appropriated enzyme may provide a unique therapeutic target to inhibit cancer progression.

These observations linking citric acid cycle enzymes to cancer

suggest that cancer is also a metabolic disease, not simply a disease of mutant growth factors and cell cycle control proteins. The realization that there is a metabolic component to cancer opens the door to new thinking about the control of cancer. Indeed, preliminary experiments suggest that if cancer cells undergoing aerobic glycolysis are forced by pharmacological manipulation to use oxidative phosphorylation, the cancer cells lose their malignant properties. It is also interesting to note that the citric acid cycle, which has been studied for decades, still has secrets to be revealed by future biochemists.

17.4 The Citric Acid Cycle Is a Source of Biosynthetic Precursors

Thus far, discussion has focused on the citric acid cycle as the major degradative pathway for the generation of ATP. As a major metabolic hub of the cell, the citric acid cycle also integrates many of the cell’s other metabolic pathways, including those of carbohydrates, fats, amino acids, and porphyrins. The cytoplasmic and mitochondrial pools of citric acid cycle components are interchangeable. This integration allows the use of citric acid intermediates for biosyntheses (Figure 17.18). For example, most of the carbon atoms in porphyrins come from succinyl CoA in a pathway that occurs both in the cytoplasm and mitochondria. Fats are synthesized in the cytoplasm from mitochondrial citrate. Many of the amino acids, used throughout the cell, are derived from α- ketoglutarate and oxaloacetate. These biosynthetic processes will be considered in subsequent chapters.

The citric acid cycle must be capable of being rapidly replenished

An important consideration is that citric acid cycle intermediates must be replenished if any are drawn off for biosyntheses. Suppose that much oxaloacetate is converted into amino acids for protein synthesis and, subsequently, the energy needs of the cell rise. The citric acid cycle will operate to a reduced extent unless new oxaloacetate is formed because acetyl CoA cannot enter the cycle unless it condenses with oxaloacetate. Even though oxaloacetate is recycled, a minimal level must be maintained to allow the cycle to function.

How is oxaloacetate replenished? Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate. Rather, oxaloacetate is formed by the carboxylation of pyruvate, in a reaction catalyzed by the biotin-dependent enzyme pyruvate carboxylase (Figure 17.19).

Pyruvate + CO2+ATP+H2O→oxaloacetate+ADP+Pi+2H+

Oxaloacetate is replenished by its formation from pyruvate. Acetyl CoA may be produced from the metabolism of both pyruvate and fatty acids.

EXPLORE this pathway further in the Metabolic Map on

Recall that this enzyme plays a crucial role in gluconeogenesis (Section 16.3). It is active only in the presence of acetyl CoA, which signifies the need for more oxaloacetate. If the energy charge is high, oxaloacetate is converted into glucose. If the energy charge is low, oxaloacetate replenishes the citric acid cycle. The synthesis of oxaloacetate by the carboxylation of pyruvate is an example of ananaplerotic reaction (anaplerotic is of Greek origin, meaning to “fill up”), a reaction that leads to the net synthesis, or replenishment, of pathway components. Note that because the citric acid cycle is a cycle, it can be replenished by the generation of any of the intermediates. Glutamine is an especially important source of citric acid cycle intermediates in rapidly growing cells, including cancer cells. Glutamine is converted into glutamate and then into α-ketoglutarate
(Section 23.5).

The disruption of pyruvate metabolism is the cause of beriberi and poisoning by mercury and arsenic

Beriberi, a neurologic and cardiovascular disorder, is caused by a

dietary deficiency of thiamine (also called vitamin B1
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. The problem is exacerbated if the rice is polished to remove the outer layer (that is, converted from brown to white rice), because only the outer layer contains significant amounts of thiamine. A form of beriberi, called Wernicke’s encephalopathy, is also occasionally seen in alcoholics who are severely malnourished and thus thiamine deficient. The disease is characterized by neurologic 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.

Beriberi

A vitamin-deficiency disease first described in 1630 by Jacob Bonitus, a Dutch physician working in Java:

“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.”

). The disease

Which biochemical processes might be affected by a deficiency of thiamine? Thiamine is the precursor of the cofactor thiamine pyrophosphate. This cofactor is the prosthetic group of three important enzymes: pyruvate dehydrogenase, α-ketoglutarate

dehydrogenase, and transketolase. Transketolase functions in the pentose phosphate pathway, which will be considered in Chapter 20. The common feature of enzymatic reactions utilizing TPP is the transfer of an activated aldehyde unit. In beriberi, the levels of pyruvate and α- ketoglutarate in the blood 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 and α-ketoglutarate dehydrogenase complexes 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 materials have a high affinity for neighboring (vicinal) sulfhydryls, such as those in the reduced dihydrolipoyl groups of the E3 component of the pyruvate dehydrogenase complex

(Figure 17.20). 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 hatmakers who used mercury nitrate to soften and shape animal furs. 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 (Figure 17.20) 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.

The manuscript proposing the citric acid cycle was submitted for publication to Nature but was rejected in June 1937. That same year it was published in Enzymologia. Dr. Krebs proudly displayed the rejection letter throughout his career as encouragement for young scientists:

“The editor of NATURE presents his compliments to Dr. H. A. Krebs and regrets that as he has already sufficient letters to fill the correspondence columns of NATURE for seven or eight weeks, it is undesirable to accept further letters at the present time on account of the time delay which must occur in their publication.

If Dr. Krebs does not mind much delay the editor is prepared to keep the letter until the congestion is relieved in the hope of making use of it.

He returns it now, in case Dr. Krebs prefers to submit it for early publication to another periodical.”

The citric acid cycle may have

evolved from preexisting pathways

How did the citric acid cycle come into being? Although definitive

answers are elusive, informed speculation is possible. We can perhaps begin to comprehend how evolution might work at the level of biochemical pathways.

The citric acid cycle was most likely assembled from preexisting reaction pathways. As noted earlier, many of the intermediates formed in the citric acid cycle are used in metabolic pathways for amino acids and porphyrins. Thus, compounds such as pyruvate, α-ketoglutarate,

and oxaloacetate were likely present early in evolution for biosynthetic purposes. The oxidative decarboxylation of these α-

ketoacids is quite favorable thermodynamically and can be used to drive the synthesis of both acyl CoA derivatives and NADH. These reactions almost certainly formed the core of processes that preceded the citric acid cycle evolutionarily. Interestingly, α-ketoglutarate

and oxaloacetate can be interconverted by transamination of the respective amino acids by aspartate

aminotransferase, another key biosynthetic enzyme. Thus, cycles comprising smaller numbers of intermediates used for a variety of biochemical purposes could have existed before the present form evolved.

17.5 The Glyoxylate Cycle Enables Plants and Bacteria to Grow on Acetate

Acetyl CoA that enters the citric acid cycle has but one fate: oxidation to CO2 and H2O. Most organisms thus cannot convert acetyl CoA

into glucose. Although oxaloacetate, a key precursor to glucose, is formed in the citric acid cycle, the two decarboxylations that take place before the regeneration of oxaloacetate preclude the net conversion of acetyl CoA into glucose.

In plants and in some microorganisms, there is a metabolic pathway that allows the conversion of acetyl CoA generated from fat stores into glucose. This reaction sequence, called the glyoxylate cycle, is similar to the citric acid cycle but bypasses the two decarboxylation steps of the cycle. Another important difference is that two molecules of acetyl CoA enter per turn of the glyoxylate cycle, compared with one in the citric acid cycle.

The glyoxylate cycle (Figure 17.21), like the citric acid cycle, begins with the condensation of acetyl CoA and oxaloacetate to form citrate, which is then isomerized to isocitrate. Instead of being decarboxylated, as in the citric acid cycle, isocitrate is cleaved by isocitrate lyase into succinate and glyoxylate. The ensuing steps regenerate oxaloacetate from glyoxylate. First, acetyl CoA condenses with glyoxylate to form malate in a reaction catalyzed by malate synthase, and then malate is oxidized to oxaloacetate, as in the citric acid cycle. The sum of these

reactions is

2 Acetyl CoA+NAD++2 H2O→ succinate + 2 CoA+NADH+2 H+

In plants, these reactions take place in organelles called glyoxysomes. This cycle is especially prominent in oil-rich seeds, such as those from sunflowers, cucumbers, and castor beans. Succinate, released midcycle, can be converted into carbohydrates by a combination of the citric acid cycle and gluconeogenesis. The carbohydrates power seedling growth until the cell can begin photosynthesis. Thus, organisms with the glyoxylate cycle gain metabolic versatility because they can use acetyl CoA as a precursor of glucose and other biomolecules. (See Biochemistry in Focus 2 [p. 567] for a discussion of the role of the glyoxylate cycle in tuberculosis.)