The tricarboxylic acid cycle (TCA cycle, also called the Krebs cycle or the citric acid cycle) plays several roles in metabolism. It is the final pathway where the oxidative metabolism of carbohydrates, amino acids, and fatty acids converge, their carbon skeletons being converted to CO2. This oxidation provides energy for the production of the majority of ATP in most animals, including humans. The cycle occurs totally in the mitochondria and is, therefore, in close proximity to the reactions of electron transport (see Electron Transport Chain), which oxidize the reduced coenzymes produced by the cycle.
The TCA cycle is an aerobic pathway, because O2 is required as the final electron acceptor. Most of the body’s catabolic pathways converge on the TCA cycle (Figure 9.1). Reactions such as the catabolism of some amino acids generate intermediates of the cycle and are called anaplerotic reactions. The citric acid cycle also supplies intermediates for a number of important synthetic reactions.
For example, the cycle functions in the formation of glucose from the carbon skeletons of some amino acids, and it provides building blocks for the synthesis of some amino acids (see Role of Folic Acid in Amino Acid Metabolism) and heme (see Biosynthesis of heme). Therefore, this cycle should not be viewed as a closed circle, but instead as a traffic circle with compounds entering and leaving as required.
Figure 9.1.The tricarboxylic acid cycle shown as a part of the central pathways of energy metabolism.
(See Figure 8.2 for a more detailed view of the metabolic map.)
Reactions of the TCA Cycle
In the TCA cycle, oxaloacetate is first condensed with an acetyl group from acetyl coenzyme A (CoA), and then is regenerated as the cycle is completed (Figure 9.1). Thus, the entry of one acetyl CoA into one round of the TCA cycle does not lead to the net production or consumption of intermediates. [Note: Two carbons entering the cycle as acetyl CoA are balanced by two CO2 exiting.]
A. Oxidative decarboxylation of pyruvate
Pyruvate, the endproduct of aerobic glycolysis, must be transported into the mitochondrion before it can enter the TCA cycle. This is accomplished by a specific pyruvate transporter that helps pyruvate cross the inner mitochondrial membrane. Once in the matrix, pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase complex, which is a multienzyme complex. Strictly speaking, the pyruvate dehydrogenase complex is not part of the TCA cycle proper, but is a major source of acetyl CoA—the two-carbon substrate for the cycle.
1. Component enzymes
The pyruvate dehydrogenase complex (PDH complex) is a multimolecular aggregate of three enzymes, pyruvate dehydrogenase (PDH or E1, also called a decarboxylase), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Each catalyzes a part of the overall reaction (Figure 9.2).
Their physical association links the reactions in proper sequence without the release of intermediates. In addition to the enzymes participating in the conversion of pyruvate to acetyl CoA, the complex also contains two tightly bound regulatory enzymes, pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.
Figure 9.2.Mechanism of action of the pyruvate dehydrogenase complex.
TPP = thiamine pyrophosphate; L = lipoic acid.
The PDH complex contains five coenzymes that act as carriers or oxidants for the intermediates of the reactions shown in Figure 9.2. E1 requires thiamine pyrophosphate (TPP), E2 requires lipoic acid and CoA, and E3 requires FAD and NAD+.
Deficiencies of thiamine or niacin can cause serious central nervous system problems. This is because brain cells are unable to produce sufficient ATP (via the TCA cycle) if the PDH complex is inactive. Wernicke-Korsakoff, an encephalopathy-psychosis syndrome due to thiamine deficiency, may be seen with alcohol abuse
3. Regulation of the pyruvate dehydrogenase complex
Covalent modification by the two regulatory enzymes that are part of the complex alternately activate and inactivate E1 (PDH). The cyclic AMP-independent PDH kinase phosphorylates and, thereby, inhibits E1, whereas PDH phosphatase dephosphorylates and activates E1 (Figure 9.3). The kinase itself is allosterically activated by ATP, acetyl CoA, and NADH. Therefore, in the presence of these high-energy signals, the PDH complex is turned off. Pyruvate is a potent inhibitor of PDH kinase.
Therefore, if pyruvate concentrations are elevated, E1 will be maximally active. Calcium is a strong activator of PDH phosphatase, stimulating E1 activity. This is particularly important in skeletal muscle, where release of Ca2+ during contraction stimulates the PDH complex, and thereby energy production. [Note: Although covalent regulation by the kinase and phosphatase is key, the complex is also subject to product (NADH, acetyl CoA) inhibition.]
Figure 9.3.Regulation of pyruvate dehydrogenase complex.
[ denotes product inhibition.]
4. Pyruvate dehydrogenase deficiency
A deficiency in the E1 component of the PDH complex, although rare, is the most common biochemical cause of congenital lactic acidosis. This enzyme deficiency results in an inability to convert pyruvate to acetyl CoA, causing pyruvate to be shunted to lactic acid via lactate dehydrogenase (see Reduction of pyruvate to lactate). This causes particular problems for the brain, which relies on the TCA cycle for most of its energy, and is particularly sensitive to acidosis.
Symptoms are variable and include neurodegeneration, muscle spasticity and, in the neonatal onset form, early death. The E1 defect is X-linked, but because of the importance of the enzyme in the brain, it affects both males and females. Therefore, the defect is classified as X-linked dominant. There is no proven treatment for pyruvate dehydrogenase deficiency: however, dietary restriction of carbohydrate and supplementation with TPP may reduce symptoms in select patients.
Leigh syndrome (subacute necrotizing encephalomyelopathy) is a rare, progressive neurological disorder that is the result of defects in mitochondrial ATP production, primarily as a result of mutations in the PDH complex, the electron transport chain, or ATP synthase. Both nuclear and mtDNA can be affected
5. Mechanism of arsenic poisoning
As previously described (see Mechanism of arsenic poisoning), arsenic can interfere with glycolysis at the glyceraldehyde 3-phosphate step, thereby decreasing ATP production. “Arsenic poisoning” is, however, due primarily to inhibition of enzymes that require lipoic acid as a coenzyme, including E2 of the PDH complex, ?-ketoglutarate dehydrogenase (see below), and branched-chain ?-keto acid dehydrogenase (see Catabolism of the branched-chain amino acids).
Arsenite (the trivalent form of arsenic) forms a stable complex with the thiol (?SH) groups of lipoic acid, making that compound unavailable to serve as a coenzyme. When it binds to lipoic acid in the PDH complex, pyruvate (and consequently lactate) accumulates. Like pyruvate dehydrogenase deficiency, this particularly affects the brain, causing neurologic disturbances and death.
B. Synthesis of citrate from acetyl CoA and oxaloacetate
The condensation of acetyl CoA and oxaloacetate to form citrate (a tricarboxylic acid) is catalyzed by citrate synthase (Figure 9.4). This aldol condensation has an equilibrium far in the direction of citrate synthesis. In humans, citrate synthase is not an allosteric enzyme. It is inhibited by its product, citrate. Substrate availability is another means of regulation for citrate synthase.
The binding of oxaloacetate causes a conformational change in the enzyme that generates a binding site for acetyl CoA. [Note: Citrate, in addition to being an intermediate in the TCA cycle, provides a source of acetyl CoA for the cytosolic synthesis of fatty acids (see De Novo Synthesis of Fatty Acids). Citrate also inhibits phosphofructokinase, the rate-limiting enzyme of glycolysis (see Isomerization of glucose 6-phosphate), and activates acetyl CoA carboxylase (the rate-limiting enzyme of fatty acid synthesis; see De Novo Synthesis of Fatty Acids).]
Figure 9.4.Formation of ?-ketoglutarate from acetyl CoA and oxaloacetate.
C. Isomerization of citrate
Citrate is isomerized to isocitrate by aconitase, an Fe-S protein (see Figure 9.4). [Note: Aconitase is inhibited by fluoroacetate, a compound that is used as a rat poison. Fluoroacetate is converted to fluoroacetyl CoA, which condenses with oxaloacetate to form fluorocitrate—a potent inhibitor of aconitase—resulting in citrate accumulation.]
D. Oxidation and decarboxylation of isocitrate
Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate, yielding the first of three NADH molecules produced by the cycle, and the first release of CO2 (see Figure 9.4). This is one of the rate-limiting steps of the TCA cycle. The enzyme is allosterically activated by ADP (a low-energy signal) and Ca2+, and is inhibited by ATP and NADH, whose levels are elevated when the cell has abundant energy stores.
E. Oxidative decarboxylation of ?-ketoglutarate
The conversion of ?-ketoglutarate to succinyl CoA is catalyzed by the ?-ketoglutarate dehydrogenase complex, a multimolecular aggregate of three enzymes (Figure 9.5). The mechanism of this oxidative decarboxylation is very similar to that used for the conversion of pyruvate to acetyl CoA by the PDH complex. The reaction releases the second CO2 and produces the second NADH of the cycle. The coenzymes required are thiamine pyrophosphate, lipoic acid, FAD, NAD+, and CoA.
Each functions as part of the catalytic mechanism in a way analogous to that described for the PDH complex (see Component enzymes). The equilibrium of the reaction is far in the direction of succinyl CoA—a high-energy thioester similar to acetyl CoA. ?-Ketoglutarate dehydrogenase complex is inhibited by its products, NADH and succinyl CoA, and activated by Ca2+. However, it is not regulated by phosphorylation/dephosphorylation reactions as described for PDH complex. [Note: ?-Ketoglutarate is also produced by the oxidative deamination (see Glutamate dehydrogenase: the oxidative deamination of amino acids) or transamination of the amino acid, glutamate (see Transamination: the funneling of amino groups to glutamate).]
Figure 9.5.Formation of malate from ?-ketoglutarate.
F. Cleavage of succinyl CoA
Succinate thiokinase (also called succinyl CoA synthetase—named for the reverse reaction) cleaves the high-energy thioester bond of succinyl CoA (see Figure 9.5). This reaction is coupled to phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). GTP and ATP are energetically interconvertible by the nucleoside diphosphate kinase reaction:
The generation of GTP by succinate thiokinase is another example of substrate-level phosphorylation (see Formation of pyruvate producing ATP). [Note: Succinyl CoA is also produced from propionyl CoA derived from the metabolism of fatty acids with an odd number of carbon atoms (see Oxidation of fatty acids with an odd number of carbons), and from the metabolism of several amino acids (see Other amino acids that form succinyl CoA).]
G. Oxidation of succinate
Succinate is oxidized to fumarate by succinate dehydrogenase, as FAD (its coenzyme) is reduced to FADH2 (see Figure 9.5). Succinate dehydrogenase is the only enzyme of the TCA cycle that is embedded in the inner mitochondrial membrane. As such, it functions as Complex II of the electron transport chain (see Coenzyme Q). [Note: FAD, rather than NAD+, is the electron acceptor because the reducing power of succinate is not sufficient to reduce NAD+.]
H. Hydration of fumarate
Fumarate is hydrated to malate in a freely reversible reaction catalyzed by fumarase (also called fumarate hydratase, see Figure 9.5). [Note: Fumarate is also produced by the urea cycle (see Figure 19.14), in purine synthesis (see Figure 22.7), and during catabolism of the amino acids, phenylalanine and tyrosine (see Phenylalanine and tyrosine).]
I. Oxidation of malate
Malate is oxidized to oxaloacetate by malate dehydrogenase (Figure 9.6). This reaction produces the third and final NADH of the cycle. The ?G0 of the reaction is positive, but the reaction is driven in the direction of oxaloacetate by the highly exergonic citrate synthase reaction. [Note: Oxaloacetate is also produced by the transamination of the amino acid, aspartic acid (see Transamination: the funneling of amino groups to glutamate).]
Figure 9.6.Formation of oxaloacetate from malate.
Energy Produced by the TCA Cycle
Two carbon atoms enter the cycle as acetyl CoA and leave as CO2. The cycle does not involve net consumption or production of oxaloacetate or of any other intermediate. Four pairs of electrons are transferred during one turn of the cycle: three pairs of electrons reducing three NAD+ to NADH and one pair reducing FAD to FADH2. Oxidation of one NADH by the electron transport chain leads to formation of approximately three ATP, whereas oxidation of FADH2 yields approximately two ATP (see ?G° of ATP). The total yield of ATP from the oxidation of one acetyl CoA is shown in Figure 9.7. Figure 9.8 summarizes the reactions of the TCA cycle.
Figure 9.7.Number of ATP molecules produced from the oxidation of one molecule of acetyl CoA (using both substrate-level and oxidative phosphorylation).
A.Production of reduced coenzymes, ATP, and CO2 in the citric acid cycle. B.Inhibitors and activators of the cycle.
Regulation of the TCA Cycle
In contrast to glycolysis, which is regulated primarily by phosphofructo-kinase, the TCA cycle is controlled by the regulation of several enzyme activities (see Figure 9.8). The most important of these regulated enzymes are those that catalyze reactions with highly negative ?G0: citrate synthase, isocitrate dehydrogenase, and ?-ketoglutarate dehydrogenase complex. Reducing equivalents needed for oxidative phosphorylation are generated by the pyruvate dehydrogenase complex and the TCA cycle, and both processes are upregulated in response to a rise in ADP.
Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase (PDH) complex, producing acetyl CoA, which is the major fuel for the tricarboxylic acid cycle (TCA cycle, Figure 9.9). This multienzyme complex requires five coenzymes: thiamine pyrophosphate, lipoic acid, FAD, NAD+, and coenzyme A. PDH complex is regulated by covalent modification of E1 (pyruvate dehydrogenase, PDH) by PDH kinase and PDH phosphatase: phosphorylation inhibits PDH. PDH kinase is allosterically activated by ATP, acetyl CoA, and NADH and inhibited by pyruvate; the phosphatase is activated by Ca2+.
Pyruvate dehydrogenase deficiency is the most common biochemical cause of congenital lactic acidosis. The central nervous system is particularly affected in this is X-linked dominant disorder. Arsenic poisoning causes inactivation of PDH complex by binding to lipoic acid. Citrate is synthesized from oxaloacetate and acetyl CoA by citrate synthase. This enzyme is subject to product inhibition by citrate. Citrate is isomerized to isocitrate by aconitase. Isocitrate is oxidized and decarboxylated by isocitrate dehydrogenase to ?-ketoglutarate, producing CO2 and NADH. The enzyme is inhibited by ATP and NADH, and activated by ADP and Ca2+.
?-Ketoglutarate is oxidatively decarboxylated to succinyl CoA by the ?-ketoglutarate dehydrogenase complex, producing CO2 and NADH. The enzyme is very similar to pyruvate dehydrogenase and uses the same coenzymes. ?-Ketoglutarate dehydrogenase complex is activated by calcium and inhibited by NADH and succinyl CoA, but is not covalently regulated. Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and GTP. This is an example of substrate-level phosphorylation. Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2. Fumarate is hydrated to malate by fumarase (fumarate hydratase), and malate is oxidized to oxaloacetate by malate dehydrogenase, producing NADH.
Three NADH, one FADH2, and one GTP (whose terminal phosphate can be transferred to ADP by nucleoside diphosphate kinase, producing ATP) are produced by one round of the TCA cycle. The generation of acetyl CoA by the oxidation of pyruvate via the PDH complex also produces an NADH. Oxidation of these NADHs and FADH2 by the electron transport chain yields 14 ATP. An additional ATP (GTP) comes from substrate level phosphorylation in the TCA cycle. Therefore, a total of 15 ATPs are produced from the complete mitochondrial oxidation of pyruvate to CO2.