Harper’s Illustrated Biochemistry, 29th Edition (2012)

Chapter 17. The Citric Acid Cycle: The Catabolism of Acetyl-CoA

David A. Bender, PhD & Peter A. Mayes, PhD, DSc

OBJECTIVES

After studying this chapter, you should be able to:

Describe the reactions of the citric acid cycle and the reactions that lead to the production of reducing equivalents that are oxidized in the mitochondrial electron transport chain to yield ATP.

Explain the importance of vitamins in the citric acid cycle.

Explain how the citric acid cycle provides both a route for catabolism of amino acids and also a route for their synthesis.

Describe the main anaplerotic pathways that permit replenishment of citric acid cycle intermediates, and how the withdrawal of oxaloacetate for gluconeogenesis is controlled.

Describe the role of the citric acid cycle in fatty acid synthesis.

Explain how the activity of the citric acid cycle is controlled by the availability of oxidized cofactors.

Explain how hyperammonemia can lead to loss of consciousness.

BIOMEDICAL IMPORTANCE

The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) is a sequence of reactions in mitochondria that oxidizes the acetyl moiety of acetyl-CoA and reduces coenzymes that are reoxidized through the electron transport chain, linked to the formation of ATP.

The citric acid cycle is the final common pathway for the oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of the cycle. It also has a central role in gluconeogenesis, lipogenesis, and interconversion of amino acids. Many of these processes occur in most tissues, but liver is the only tissue in which all occur to a significant extent. The repercussions are therefore profound when, for example, large numbers of hepatic cells are damaged as in acute hepatitis or replaced by connective tissue (as in cirrhosis). The few genetic defects of citric acid cycle enzymes that have been reported are associated with severe neurological damage as a result of very considerably impaired ATP formation in the central nervous system.

Hyperammonemia, as occurs in advanced liver disease, leads to loss of consciousness, coma, and convulsions as a result of impaired activity of the citric acid cycle, leading to reduced formation of ATP. Ammonia both depletes citric acid cycle intermediates (by withdrawing α-ketoglutarate for the formation of glutamate and glutamine) and also inhibits the oxidative decarboxylation of α-ketoglutarate.

THE CITRIC ACID CYCLE PROVIDES SUBSTRATE FOR THE RESPIRATORY CHAIN

The cycle starts with reaction between the acetyl moiety of acetyl-CoA and the four-carbon dicarboxylic acid oxaloacetate, forming a six-carbon tricarboxylic acid, citrate. In the subsequent reactions, two molecules of CO₂ are released and oxaloacetate is regenerated (Figure 17–1). Only a small quantity of oxaloacetate is needed for the oxidation of a large quantity of acetyl-CoA; it can be considered as playing a catalytic role, since it is regenerated at the end of the cycle.

FIGURE 17–1 The citric acid cycle, illustrating the catalytic role of oxaloacetate.

The citric acid cycle is an integral part of the process by which much of the free energy liberated during the oxidation of fuels is made available. During the oxidation of acetyl-CoA, coenzymes are reduced and subsequently reoxidized in the respiratory chain, linked to the formation of ATP (oxidative phosphorylation, Figure 17–2; see also Chapter 13). This process is aerobic, requiring oxygen as the final oxidant of the reduced coenzymes. The enzymes of the citric acid cycle are located in the mitochondrial matrix, either free or attached to the inner mitochondrial membrane and the crista membrane, where the enzymes and coenzymes of the respiratory chain are also found (Chapter 13).

FIGURE 17–2 The citric acid cycle: the major catabolic pathway for acetyl-CoA in aerobic organisms. Acetyl-CoA, the product of carbohydrate, protein, and lipid catabolism, is taken into the cycle and oxidized to CO₂ with the release of reducing equivalents (2H). Subsequent oxidation of 2H in the respiratory chain leads to phosphorylation of ADP to ATP. For one turn of the cycle, nine ATP are generated via oxidative phosphorylation and one ATP (or GTP) arises at substrate level from the conversion of succinyl-CoA to succinate.

REACTIONS OF THE CITRIC ACID CYCLE LIBERATE REDUCING EQUIVALENTS & CO₂

The initial reaction between acetyl-CoA and oxaloacetate to form citrate is catalyzed by citrate synthase, which forms a carboncarbon bond between the methyl carbon of acetyl-CoA and the carbonyl carbon of oxaloacetate (Figure 17–3). The thioester bond of the resultant citryl-CoA is hydrolyzed, releasing citrate and CoASH—an exothermic reaction.

FIGURE 17–3 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 two carbon atoms of the acetyl radical are shown labeled on the carboxyl carbon (*) and on the methyl carbon (•). Although two carbon atoms are lost as CO₂ in one 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 four carbon atoms of oxaloacetate appear to be labeled after one turn of the cycle. During gluconeogenesis, some of the label in oxaloacetate is incorporated into glucose and glycogen (Figure 20–1). The sites of inhibition () by fluoroacetate, malonate, and arsenite are indicated.

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase); the reaction occurs in two steps: dehydration to cis-aconitate and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is the result of channeling—transfer of the product of citrate synthase directly onto the active site of aconitase, without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. Citrate is only available in free solution to be transported from the mitochondria to the cytosol for fatty acid synthesis when aconitase is inhibited by accumulation of its product, isocitrate.

The poison fluoracetate is found in some of plants, and their consumption can be fatal to grazing animals. Some fluorinated compounds used as anticancer agents and industrial chemicals (including pesticides) are metabolized to fluoroacetate. It is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate.

Isocitrate undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form, initially, oxalosuccinate, which remains enzyme bound and undergoes decarboxylation to α-ketoglutarate. The decarboxylation requires Mg²⁺or Mn²⁺ ions. There are three isoenzymes of isocitrate dehydrogenase. One, which uses NAD⁺, is found only in mitochondria. The other two use NADP⁺ and are found in mitochondria and the cytosol. Respiratory-chain-linked oxidation of isocitrate occurs through the NAD⁺-dependent enzyme.

α-Ketoglutarate undergoes oxidative decarboxylation in a reaction catalyzed by a multienzyme complex similar to that involved in the oxidative decarboxylation of pyruvate (Figure 18–5). The α-ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex—thiamin diphosphate, lipoate, NAD⁺, FAD, and CoA—and results in the formation of succinyl-CoA. The equilibrium of this reaction is so much in favor of succinyl-CoA formation that it must be considered to be physiologically unidirectional. As in the case of pyruvate oxidation (Chapter 18), arsenite inhibits the reaction, causing the substrate, α-ketoglutarate, to accumulate. High concentrations of ammonia inhibit α-ketoglutarate dehydrogenase.

Succinyl-CoA is converted to succinate by the enzyme succinate thiokinase (succinyl-CoA synthetase). This is the only example of substrate level phosphorylation in the citric acid cycle. Tissues in which gluconeogenesis occurs (the liver and kidney) contain two isoenzymes of succinate thiokinase, one specific for GDP and the other for ADP. The GTP formed is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis, and provides a regulatory link between citric acid cycle activity and the withdrawal of oxaloacetate for gluconeogenesis. Nongluconeogenic tissues have only the isoenzyme that uses ADP.

When ketone bodies are being metabolized in extrahepatic tissues, there is an alternative reaction catalyzed by succinyl-CoA-acetoacetate-CoA transferase (thiophorase), involving transfer of CoA from succinyl-CoA to acetoacetate, forming acetoacetyl-CoA and succinate (Chapter 22).

The onward metabolism of succinate, leading to the regeneration of oxaloacetate, is the same sequence of chemical reactions as occurs in the β-oxidation of fatty acids: dehydrogenation to form a carbon-carbon double bond, addition of water to form a hydroxyl group, and a further dehydrogenation to yield the oxo-group of oxaloacetate.

The first dehydrogenation reaction, forming fumarate, is catalyzed by succinate dehydrogenase, which is bound to the inner surface of the inner mitochondrial membrane. The enzyme contains FAD and ironsulfur (Fe:S) protein, and directly reduces ubiquinone in the electron transport chain. Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate. Malate is converted to oxaloacetate by malate dehydrogenase, a reaction requiring NAD⁺. Although the equilibrium of this reaction strongly favors malate, the net flux is to oxaloacetate because of the continual removal of oxaloacetate (to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to aspartate) and also the continual reoxidation of NADH.

TEN ATP ARE FORMED PER TURN OF THE CITRIC ACID CYCLE

As a result of oxidations catalyzed by the dehydrogenases of the citric acid cycle, three molecules of NADH and one of FADH₂ are produced for each molecule of acetyl-CoA catabolized in one turn of the cycle. These reducing equivalents are transferred to the respiratory chain (see Figure 13–3), where reoxidation of each NADH results in formation of ~2.5 ATP, and of FADH², ~1.5 ATP. In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation catalyzed by succinate thiokinase.

VITAMINS PLAY KEY ROLES IN THE CITRIC ACID CYCLE

Four of the B vitamins (Chapter 44) are essential in the citric acid cycle and hence energy-yielding metabolism: (1) riboflavin, in the form of flavin adenine dinucleotide (FAD), a cofactor for succinate dehydrogenase; (2) niacin, in the form of nicotinamide adenine dinucleotide (NAD), the electron acceptor for isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase; (3) thiamin (vitamin B₁), as thiamin diphosphate, the coenzyme for decarboxylation in the α-ketoglutarate dehydrogenase reaction; and (4) pantothenic acid, as part of coenzyme A, the cofactor attached to “active” carboxylic acid residues such as acetyl-CoA and succinyl-CoA.

THE CITRIC ACID CYCLE PLAYS A PIVOTAL ROLE IN METABOLISM

The citric acid cycle is not only a pathway for oxidation of two carbon units, but is also a major pathway for interconversion of metabolites arising from transamination and deamination of amino acids (Chapters 28 & 29), and providing the substrates for amino acid synthesis by transamination (Chapter 27), as well as for gluconeogenesis (Chapter 20) and fatty acid synthesis (Chapter 23). Because it functions in both oxidative and synthetic processes, it is amphibolic (Figure 17–4).

FIGURE 17–4 Involvement of the citric acid cycle in transamination and gluconeogenesis. The bold arrows indicate the main pathway of gluconeogenesis.

The Citric Acid Cycle Takes Part in Gluconeogenesis, Transamination, & Deamination

All the intermediates of the cycle are potentially glucogenic, since they can give rise to oxaloacetate, and hence net production of glucose (in the liver and kidney, the organs that carry out gluconeogenesis; see Chapter 20). The key enzyme that catalyzes net transfer out of the cycle into gluconeogenesis is phosphoenolpyruvate carboxykinase, which catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate, with GTP acting as the phosphate donor (see Figure 20–1). The GTP required for this reaction is provided (in liver and kidney) by the GDP-dependent isoenzyme of succinate thiokinase. This ensures that oxaloacetate will not be withdrawn from the cycle for gluconeogenesis if this would lead to depletion of citric acid cycle intermediates, and hence reduced generation of ATP.

Net transfer into the cycle occurs as a result of several reactions. Among the most important of such anaplerotic reactions is the formation of oxaloacetate by the carboxylation of pyruvate, catalyzed by pyruvate carboxylase.This reaction is important in maintaining an adequate concentration of oxaloacetate for the condensation reaction with acetyl-CoA. If acetyl-CoA accumulates, it acts as both an allosteric activator of pyruvate carboxylase and an inhibitor of pyruvate dehydrogenase, thereby ensuring a supply of oxaloacetate. Lactate, an important substrate for gluconeogenesis, enters the cycle via oxidation to pyruvate and then carboxylation to oxaloacetate. Glutamate and glutamine are important anaplerotic substrates because they yield α-ketoglutarate as a result of the reactions catalyzed by glutaminase and glutamate dehydrogenase. Transamination of aspartate leads directly to the formation of oxaloacetate, and a variety of compounds that are metabolized to yield propionyl CoA, which can be carboxylated and isomerized to succinyl CoA are also important anaplerotic substrates.

Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from aspartate, and α-ketoglutarate from glutamate. Because these reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of these amino acids. Other amino acids contribute to gluconeogenesis because their carbon skeletons give rise to citric acid cycle intermediates. Alanine, cysteine, glycine, hydroxyproline, serine, threonine, and tryptophan yield pyruvate; arginine, histidine, glutamine, and proline yield α-ketoglutarate; isoleucine, methionine, and valine yield succinyl-CoA; tyrosine and phenylalanine yield fumarate (see Figure 17–4).

The citric acid cycle itself does not provide a pathway for the complete oxidation of the carbon skeletons of amino acids that give rise to intermediates such as α-ketoglutarate, succinyl CoA, fumarate and oxaloacetate, because this results in an increase in the amount of oxaloacetate. For complete oxidation to occur, oxaloacetate must undergo phosphorylation and carboxylation to phosphoenolpyruvate (at the expense of GTP) then dephosphorylation to pyruvate (catalyzed by pyruvate kinase) and oxidative decarboxylation to acetyl Co (catalyzed by pyruvate dehydrogenase).

In ruminants, whose main metabolic fuel is short-chain fatty acids formed by bacterial fermentation, the conversion of propionate, the major glucogenic product of rumen fermentation, to succinyl-CoA via the methylmalonyl-CoA pathway (Figure 20–2) is especially important.

The Citric Acid Cycle Takes Part in Fatty Acid Synthesis

Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, is the major substrate for long-chain fatty acid synthesis in nonruminants (Figure 17–5). (In ruminants, acetyl-CoA is derived directly from acetate.) Pyruvate dehydrogenase is a mitochondrial enzyme, and fatty acid synthesis is a cytosolic pathway; the mitochondrial membrane is impermeable to acetyl-CoA. Acetyl-CoA is made available in the cytosol from citrate synthesized in the mitochondrion, transported into the cytosol, and cleaved in a reaction catalyzed by ATP-citrate lyase (Figure 17–5). Citrate is only available for transport out of the mitochondrion when aconitase is inhibted by its product and therefore saturated with its substrate, so that citrate cannot be channeled directly from citrate synthase onto aconitase. This ensures that citrate is used for fatty acid synthesis only when there is an adequate amount to ensure continued activity of the cycle.

FIGURE 17–5 Participation of the citric acid cycle in fatty acid synthesis from glucose. See also Figure 23–5.

Regulation of the Citric Acid Cycle Depends Primarily on a Supply of Oxidized Cofactors

In most tissues, where the primary role of the citric acid cycle is in energy-yielding metabolism, respiratory control via the respiratory chain and oxidative phosphorylation regulates citric acid cycle activity (Chapter 13). Thus, activity is immediately dependent on the supply of NAD⁺, which in turn, because of the tight coupling between oxidation and phosphorylation, is dependent on the availability of ADP and hence, ultimately on the rate of utilization of ATP in chemical and physical work. In addition, individual enzymes of the cycle are regulated. The most likely sites for regulation are the nonequilibrium reactions catalyzed by pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. The dehydrogenases are activated by Ca²+, which increases in concentration during contraction of muscle and secretion by other tissues, when there is increased energy demand. In a tissue such as brain, which is largely dependent on carbohydrate to supply acetyl-CoA, control of the citric acid cycle may occur at pyruvate dehydrogenase. Several enzymes are responsive to the energy status as shown by the [ATP]/[ADP] and [NADH]/[NAD⁺] ratios. Thus, there is allosteric inhibition of citrate synthase by ATP and long-chain fatty acyl-CoA. Allosteric activation of mitochondrial NAD-dependent isocitrate dehydrogenase by ADP is counteracted by ATP and NADH. The α-ketoglutarate dehydrogenase complex is regulated in the same way as is pyruvate dehydrogenase (Figure 18–6). Succinate dehydrogenase is inhibited by oxaloacetate, and the availability of oxaloacetate, as controlled by malate dehydrogenase, depends on the [NADH]/[NAD⁺] ratio. Since the K for oxaloacetate of citrate synthase is of the same order of magnitude as the intramitochondrial concentration, it is likely that the concentration of oxaloacetate controls the rate of citrate formation. Which of these mechanisms are important in vivo is still to be resolved.

Hyperammonemia, as occurs in advanced liver disease and a number of (rare) genetic diseases of amino acid metabolism, leads to loss of consciousness, coma and convulsions, and may be fatal. This is because of the withdrawal of α-ketoglutarate to form glutamate (catalyzed by glutamate dehydrogenase) and then glutamine (catalyzed by glutamine synthetase), leading to reduced concentrations of all citric acid cycle intermediates, and hence reduced generation of ATP. The equilibrium of glutamate dehydrogenase is finely poised, and the direction of reaction depends on the ratio of NAD⁺ : NADH and the concentration of ammonium ions. In addition, ammonia inhibits α-ketoglutarate dehydrogenase, and possibly also pyruvate dehydrogenase.

SUMMARY

The citric acid cycle is the final pathway for the oxidation of carbohydrate, lipid, and protein. Their common end-metabolite, acetyl-CoA, reacts with oxaloacetate to form citrate. By a series of dehydrogenations and decarboxylations, citrate is degraded, reducing coenzymes, releasing 2 CO₂, and regenerating oxaloacetate.

The reduced coenzymes are oxidized by the respiratory chain linked to formation of ATP. Thus, the cycle is the major pathway for the formation of ATP and is located in the matrix of mitochondria adjacent to the enzymes of the respiratory chain and oxidative phosphorylation.

The citric acid cycle is amphibolic, since in addition to oxidation it is important in the provision of carbon skeletons for gluconeogenesis, fatty acid synthesis, and interconversion of amino acids.

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