CHAPTER OUTLINE
Oxidative Decarboxylation of Pyruvate
The Pyruvate Dehydrogenase Complex
Citrate Synthase (Condensing Enzyme)
α-Ketoglutarate Dehydrogenase Complex
Succinyl-CoA Synthetase (Succinate-CoA Ligase, Succinate Thiokinase)
High-Yield Terms
PDH kinases: allosteric regulated family of kinases that modify the activity of the PDHc
Malate-aspartate shuttle: principal mechanism for the movement of reducing equivalents (in the form of NADH) from the cytoplasm to the mitochondria
Substrate-level phosphorylation: the succinyl-CoA synthetase–catalyzed reaction involves the use of the high-energy thioester of succinyl-CoA to drive synthesis of a high-energy nucleotide phosphate (GTP). This process is referred to as substrate-level phosphorylation
The bulk of ATP used by many cells to maintain homeostasis is produced by the oxidation of pyruvate in the TCA cycle. During this oxidation process, NADH and FADH2 are generated. The NADH and FADH2 are principally used to drive mitochondrial oxidative phosphorylation, a process for converting the reducing potential of NADH and FADH2 into the synthesis of high-energy phosphate in ATP.
Oxidative Decarboxylation of Pyruvate
The fate of pyruvate depends on the cell energy charge. In liver, intestine, and kidney under conditions of high-energy charge, pyruvate is directed toward gluconeogenesis. However, when the energy charge is low, pyruvate is preferentially oxidized to CO2 and H2O in the TCA cycle. The oxidation of the carbon atoms of pyruvate results in the generation of 15 equivalents of ATP per pyruvate. The enzymatic activities of the TCA cycle (and of oxidative phosphorylation) are located in the mitochondrion. When transported into the mitochondrion, pyruvate encounters 2 principal metabolizing enzymes: pyruvate carboxylase (PC) and the pyruvate dehydrogenase complex (PDHc).
With a high cell-energy charge, coenzyme A (CoA) is highly acylated, principally as acetyl-CoA, and able to allosterically activate PC, directing pyruvate toward gluconeogenesis (see Chapter 13). When the energy charge is low, CoA is not acylated and PC is inactive. Under these conditions, pyruvate is preferentially metabolized to CO2 and H2O via the PDHc and the enzymes of the TCA. Reduced NADH and FADH2 generated during the oxidative reactions can then be used to drive ATP synthesis via oxidative phosphorylation.
The Pyruvate Dehydrogenase Complex
The Pyruvate Dehydrogenase Complex (PDHc) is composed of multiple copies of 3 separate enzymes: pyruvate dehydrogenase (PDH, E1: 20-30 copies), dihydrolipoamide S-acetyltransferase (DLAT, E2: 60 copies), and dihydrolipoamide dehydrogenase (DLD, E3: 6 copies). The complex also requires 5 different coenzymes: CoA, NAD+, FAD+, lipoic acid, and thiamine pyrophosphate (TPP). The factors required for the function of the PDHc can be remembered by the mnemonic: Tender (thiamine) Loving (lipoate) Care (coenzyme A) For (flavin) Nancy (nicotinamide), TLCFN. Three of the coenzymes of the complex are tightly bound to enzymes of the complex (TPP, lipoic acid, and FAD+) and 2 are employed as carriers of the products of PDHc activity (CoA and NAD+) (Figure 16-1).
FIGURE 16-1: Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (FAD, flavin adenine dinucleotide; NAD+, nicotinamide adenine dinucleotide; TDP, thiamin diphosphate.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
The first enzyme of the PDHc is PDH itself (E1) which oxidatively decarboxylates pyruvate. The resulting acetyl group is initially bound to TPP then to the lipoic acid coenzyme of DLAT. The acetyl group is then transferred to CoA, forming acetyl-CoA. In the process, the electrons released during the oxidation pass from pyruvate to FAD (forming FADH2) and ultimately to NAD+ (forming NADH). The formation of NADH is catalyzed via the DLD enzymes of the PDHc. The fate of the NADH is oxidation via mitochondrial electron transport to produce 3 equivalents of ATP. The net result of the reactions of the PDHc is:
Pyruvate + CoA + NAD+ → CO2 + acetyl-CoA + NADH + H+
Regulation of the PDH Complex
The reactions of the PDHc serve to interconnect the metabolic pathways of glycolysis, gluconeogenesis, and fatty acid oxidation to the TCA cycle. The activity of the PDHc is also important in regulating the flux from glucose to malonyl-CoA, which is required for the de novo synthesis of fatty acids (see Chapter 19). The activity of the PDHc is highly regulated by a variety of allosteric effectors and by covalent modification. The importance of the PDHc to the maintenance of homeostasis is evident from the fact that although diseases associated with deficiencies of the PDHc have been observed, affected individuals often do not survive to maturity. Since metabolism in highly aerobic tissues such as the brain, skeletal muscle, and the heart is dependent on normal conversion of pyruvate to acetyl-CoA, aerobic tissues are most sensitive to deficiencies in components of the PDHc. Most genetic diseases associated with PDHc deficiency are due to mutations in PDH. The main pathologic result of such mutations is moderate-to-severe cerebral lactic acidosis and encephalopathies.
Acetyl-CoA, produced in the mitochondria via the action of the PDHc, will be transported out into the cytosol in the form of citrate when the energy charge of the cell rises. In the cytosol the citrate is hydrolyzed by ATP citrate lyase, yielding oxaloacetate and acetyl-CoA. In the cytosol the acetyl-CoA can be converted to malonyl-CoA via the action of acetyl-CoA carboxylase (ACC). This malonyl-CoA serves as the precursor for the synthesis of fatty acids. Accumulation of cytosolic malonyl-CoA, as will happen in energy-rich conditions, partitions cytosolic fatty acids away from the oxidizing machinery of the mitochondria by inhibiting carnitine palmitoyltransferase I (CPT I). This effect of malonyl-CoA, derived from acetyl-CoA, couples increased rates of glucose oxidation to inhibition of fatty acid oxidation (Figure 16-2).
FIGURE 16-2: Factors regulating the activity of the PDHc. PDH activity is regulated by its state of phosphorylation; being most active in the dephosphorylated state. Phosphorylation of PDH is catalyzed by 4 specific PDH kinases, designated PDK1, PDK2, PDK3, and PDK4. The activity of these kinases is enhanced when cellular energy charge is high, which is reflected by an increase in the level of ATP, NADH, and acetyl-CoA. Conversely, an increase in pyruvate strongly inhibits PDH kinases. Additional negative effectors of PDH kinases are ADP, NAD+, and CoASH, the levels of which increase when energy levels fall. The regulation of PDH phosphatases (PDPs) is less well understood but it is known that Mg2+ and Ca2+ activate the enzymes and that they are targets of insulin action. In adipose tissue, insulin increases PDH activity and in cardiac muscle PDH activity is increased by catecholamines. Reproduced with permission of themedicalbiochemistrypage, LLC.
The activity of PDH (E1) is rapidly regulated by phosphorylation and dephosphorylation events that are catalyzed by PDH kinases (PDKs) and PDH phosphatases (PDPs), respectively. Phosphorylation of PDH results in inhibition of activity, whereas, dephosphorylation increases it. Four PDK isozymes have been identified in humans: PDK1, PDK2, PDK3, and PDK4. Analysis of the patterns of expression of the PDKs shows that PDK2 is the most widely expressed with the highest levels of expression seen in heart, liver, and kidney. PDK1 expression is highest in the heart and is not seen in the liver. PDK4 expression is high in heart, liver, pancreatic islets, and kidney.
Regulation of the activity of the various PDKs exhibits isoform specificity. PDK2 is most sensitive to inhibition by pyruvate, whereas, PDK4 is relatively insensitive. Energy charge, reflected in ATP:ADP and NADH:NAD ratios, influence the activity of the PDKs. High ATP levels result in allosteric activation of the PDKs to ensure the excess carbon atoms can be diverted into anabolic synthesis pathways instead of into the TCA cycle. Conversely, as the ATP:ADP ratio falls, the increasing ADP will exert an allosteric inhibition on the activity of the PDKs. ADP exerts its allosteric inhibition of PDKs both independently and in synergy with pyruvate inhibition. In contrast to inhibition of PDKs by pyruvate, products of the PDHc, namely acetyl-CoA and NADH, allosterically activate the PDKs. PDK2 is the most sensitive to increases in the level of acetyl-CoA in comparison to the other PDK isoforms. The ratio of NADH to NAD+ also exert isoform-specific allosteric regulation of PDKs. PDK4 is highly activated by an increased NADH/NAD+, whereas, PDK2 is much less sensitive to this activation. NADH and acetyl-CoA are also negative allosteric effectors, the nonphosphorylated and active form of the PDHc. Both effectors reduce the affinity of the enzyme for pyruvate, thus limiting the flow of carbon through the PDHc. Note, however, that pyruvate is a potent negative effector on all PDKs, with the result that when pyruvate levels rise, active PDH will be favored even with high levels of NADH and acetyl-CoA.
Phosphate removal from the PDHc is catalyzed by 2 genetically and biochemically distinct PDP isoforms, PDP1 and PDP2. PDP1 is an Mg2+-dependent and Ca2+-stimulated protein serine phosphatase of the protein phosphatase-2C (PP-2C) superfamily (see Chapter 40) composed of a catalytic subunit (PDPc) and a regulatory subunit (PDPr). PDP1 is considered a major regulator of PDHc activity. The role of Ca2+ in stimulating the phosphatase activity of PDP1 toward the PDHc is by increasing its interaction with the PDHc. Calcium ion also increases the association of PDPc with the phosphorylated E1α subunit of PDH. Both PDP1 subunits are targeted by reactions of the TCA cycle (Figure 16-3).
FIGURE 16-3: The citric acid (Krebs) cycle. Oxidation of NADH and FADH2 in the respiratory chain leads to the formation of ATP via oxidative phosphorylation. In order to follow the passage of acetyl-CoA through the cycle, the 2 carbon atoms of the acetyl radical are shown labeled on the carboxyl carbon (*) and on the methyl carbon (·). Although 2 carbon atoms are lost as CO2 in 1 turn of the cycle, these atoms are not derived from the acetyl-CoA that has immediately entered the cycle, but from that portion of the citrate molecule that was derived from oxaloacetate. However, on completion of a single turn of the cycle, the oxaloacetate that is regenerated is now labeled, which leads to labeled CO2 being evolved during the second turn of the cycle. Because succinate is a symmetric compound, “randomization” of label occurs at this step so that all 4 carbon atoms of oxaloacetate appear to be labeled after 1 turn of the cycle. During gluconeogenesis, some of the label in oxaloacetate is incorporated into glucose and glycogen. The sites of inhibition (
) by fluoroacetate, malonate, and arsenite are indicated. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Citrate Synthase (Condensing Enzyme)
The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate (OAA). The standard free energy of the reaction, −8.0 kcal/mol, propels reaction in the forward direction. Since the formation of OAA from malate is thermodynamically unfavorable, the highly exergonic nature of the citrate synthase reaction is of central importance in keeping the entire cycle going in the forward direction, since it drives oxaloacetate formation by mass action principals.
When the cellular energy charge increases, the rate of flux through the TCA cycle will decline leading to a buildup of citrate. Excess citrate is used to transport acetyl-CoA carbons from the mitochondrion to the cytoplasm, where they can be used for fatty acid and cholesterol biosynthesis. Additionally, the increased levels of citrate in the cytoplasm activate the key regulatory enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase (ACC), while also inhibiting the rate-limiting enzyme of glycolysis, PFK-1.
Aconitase
The isomerization of citrate to isocitrate by aconitase is stereospecific. The stereospecific nature of the isomerization determines that the CO2 lost, as isocitrate is oxidized to succinyl-CoA, is derived from the oxaloacetate used in citrate synthesis. Aconitase is one of several mitochondrial enzymes known as non-heme iron proteins. These proteins contain inorganic iron and sulfur, known as iron sulfur centers, in a coordination complex with cysteine sulfurs of the protein.
Isocitrate Dehydrogenase
Isocitrate is oxidatively decarboxylated to α-ketoglutarate (2-oxoglutarate) by isocitrate dehydrogenase, IDH. IDH catalyzes the rate-limiting step, as well as the first NADH-yielding reaction of the TCA cycle. Control of carbon flow through the TCA cycle is regulated at IDH by the powerful negative allosteric effectors NADH and ATP and by the potent positive effectors, isocitrate, ADP, and AMP. From this fact, it should be clear that cell energy charge is a key component in regulating carbon flow through the TCA cycle.
α-Ketoglutarate Dehydrogenase Complex
α-ketoglutarate (also called 2-oxoglutarate) is oxidatively decarboxylated to succinyl-CoA by the α-ketoglutarate dehydrogenase (AKGDH) complex. The AKGDH complex is also called 2-oxoglutarate dehydrogenase (OGDH). This reaction generates the second TCA cycle equivalent of CO2 and NADH. This multienzyme complex is very similar to the PDHc in the intricacy of its protein makeup, cofactors (TLCFN), and its mechanism of action. Also, as with the PDHc, the reactions of the AKGDH complex proceed with a large negative-standard free-energy change. Although the AKGDH complex is not subject to covalent modification, allosteric regulation is quite complex, with activity being controlled by energy charge, the NADH/NAD+ ratio, and effector activity of substrates and products.
Succinyl-CoA and α-ketoglutarate are also important metabolites outside the TCA cycle. In particular, α-ketoglutarate represents a key anaplerotic metabolite linking the entry and exit of carbon atoms from the TCA cycle to pathways involved in amino acid metabolism. α-ketoglutarate is also important for driving the malate-aspartate shuttle (see Figure 13-2). Succinyl-CoA (along with glycine) contributes all the carbon and nitrogen atoms required for heme synthesis (see Chapter 33) and for nonhepatic tissue utilization of ketone bodies (see Chapter 25).
Succinyl-CoA Synthetase (succinate-CoA ligase, succinate thiokinase)
Mitochondrial GTP (guanosine triphosphate) is used in a trans-phosphorylation reaction catalyzed by the mitochondrial enzyme nucleoside diphosphokinase to phosphorylate ADP, producing ATP and regenerating GDP for the continued operation of succinyl-CoA synthetase.
Succinate Dehydrogenase
Succinate dehydrogenase (SDH) catalyzes the oxidation of succinate to fumarate with the sequential reduction of enzyme-bound FAD to FADH2. In mammalian cells, the final electron acceptor in this reaction is coenzyme Q (CoQ) of the oxidative phosphorylation machinery.
Fumarase (Fumarate Hydratase)
The fumarase-catalyzed reaction is specific for the trans form of fumarate. The result is that the hydration of fumarate proceeds stereospecifically with the production of L-malate.
Malate Dehydrogenase
L-malate is the specific substrate for malate dehydrogenase (MDH), the final enzyme of the TCA cycle. The oxidation of malate to oxaloacetate (OAA) has a standard free energy of approximately +7 kcal/mol. This indicates that the reaction, in the TCA cycle direction, is thermodynamically unfavorable. However, as noted earlier, the citrate synthase reaction that condenses OAA with acetyl-CoA has a standard free energy of about −8 kcal/mol and is responsible for pulling the MDH reaction in the forward direction.
TCA Cycle Stoichiometry
The overall stoichiometry of the TCA cycle, beginning and ending with acetyl-CoA, can be written as:
Regulation of the TCA Cycle
Regulation of the TCA cycle, like that of glycolysis, occurs at both the level of entry of substrates into the cycle as well as at the key reactions of the cycle. Fuel enters the TCA cycle primarily as acetyl-CoA. The generation of acetyl-CoA from carbohydrates is, therefore, a major control point of the cycle. This is the reaction catalyzed by the PDHc with regulation being effected as described earlier.
