The Catabolism of AcetylCoA

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

BIOMEDICAL IMPORTANCE

The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) is a series of reactions in mitochondria that oxidize acetyl residues (as acetyl-CoA) and reduce coenzymes that upon reoxidation are linked to the formation of ATP.

The citric acid cycle is the final common pathway for the aerobic 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 the 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). Very few, if any, genetic abnormalities of citric acid cycle enzymes have been reported; such abnormalities would be incompatible with life or normal development.

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 ox-aloacetate, forming a six-carbon tricarboxylic acid, citrate. In the subsequent reactions, two molecules of CO2 are released and oxaloacetate is regenerated (Figure 16-1). Only a small quantity of oxaloacetate is needed for the oxidation of a large quantity of acetyl-CoA; ox-aloacetate may be considered to play a catalytic role.

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 oxidation of acetyl-CoA, coenzymes are reduced and subsequently reoxidized in the respiratory chain, linked to the formation of ATP (oxidative phosphorylation; see Figure 16-2 and also Chapter 12). This process is aerobic, requiring oxygen as the final oxidant of the reduced coenzymes. The enzymes of the citric acid cycle are lo cated in the mitochondrial matrix, either free or attached to the inner mitochondrial membrane, where the enzymes of the respiratory chain are also found.

REACTIONS OF THE CITRIC ACID CYCLE LIBERATE REDUCING EQUIVALENTS & CO2 (Figure 16-3)*

The initial reaction between acetyl-CoA and oxaloac-etate to form citrate is catalyzed by citrate synthase which forms a carbon-carbon bond between the methyl carbon of acetyl-CoA and the carbonyl carbon of ox-aloacetate. The thioester bond of the resultant citryl-CoA is hydrolyzed, releasing citrate and CoASH—an exergonic reaction.

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase); the reaction occurs in two steps: dehydration to «'s-aconitate, some of which remains bound to the enzyme; and rehydration to isocitrate. Although citrate is a symmetric molecule, aconi-tase 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 due to 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. The poison fluo-roacetate 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, oxalosucci-nate, which remains enzyme-bound and undergoes de-carboxylation to a-ketoglutarate. The decarboxylation

*From Circular No. 200 of the Committee of Editors of Biochemical Journals Recommendations (1975): "According to standard biochemical convention, the ending ate in, eg, palmitate, denotes any mixture of free acid and the ionized form(s) (according to pH) in which the cations are not specified." The same convention is adopted in this text for all carboxylic acids.

Acetyl-CoA (C2)

Acetyl-CoA (C2)

Oxaloacetate (C4)

Citrate (Ce)

Figure 16-1. Citric acid cycle, illustrating the catalytic role of oxaloacetate.

Figure 16-1. Citric acid cycle, illustrating the catalytic role of oxaloacetate.

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 proceeds almost completely through the NAD+-dependent enzyme.

a-Ketoglutarate undergoes oxidative decarboxylation in a reaction catalyzed by a multi-enzyme complex similar to that involved in the oxidative decarboxylation of pyruvate (Figure 17-5). The a-ketoglutarate dehy-drogenase 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 physiologically unidirectional. As in the case of pyruvate oxidation (Chapter 17), arsenite inhibits the reaction, causing the substrate, a-ketoglutarate, to accumulate.

Succinyl-CoA is converted to succinate by the enzyme succinate thiokinase (succinyl-CoA synthe-tase). This is the only example in the citric acid cycle of substrate-level phosphorylation. Tissues in which glu-coneogenesis 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 phos-phoenolpyruvate 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.

Protein

Acetyl-CoA (C2)

Citrate

Citrate

a-Ketoglutarate .(C5)

Fumarate (C4) y

Fumarate (C4) y

Isocitrate

I Succinate

a-Ketoglutarate .(C5)

Cyt c

Succinyl-CoA

Cyt c

V2O.

Oxidative ^phosphorylation

V2O.

Anaerobiosis (hypoxia, anoxia)

1 y Respiratory chain 2

Fp Flavoprotein

Cyt Cytochrome

High-energy phosphate

Figure 16-2. The citric acid cycle: the major catabo-lic pathway for acetyl-CoA in aerobic organisms. Acetyl-CoA, the product of carbohydrate, protein, and lipid ca-tabolism, is taken into the cycle, together with H2O, and oxidized to CO2 with the release of reducing equivalents (2H). Subsequent oxidation of 2H in the respiratory chain leads to coupled phosphorylation of ADP to ATP. For one turn of the cycle, 11~® are generated via oxidative phosphorylation and one ~® arises at substrate level from the conversion of succinyl-CoA to succinate.

CH2 — COO-H+ Oxaloacetate

NAD+

CH2 — COO-H+ Oxaloacetate

NAD+

Fluoroacetate

Cis-aconitate Jk

Cis-aconitate Jk e*

fadh2

SUCCINATE DEHYDROGENASE

aconitase

Succinate

Succinate

The Catabolism Release Co2

Arsenite co2

NAD+

Isocitrate nadh + h+

Arsenite

ch2 I

a-Ketoglutarate

NAD+

Figure 16-3. Reactions of the citric acid (Krebs) cycle. Oxidation of NADH and FADH2 in the respiratory chain leads to the generation 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 (designated by asterisk) and on the methyl carbon (using the designation •). Although two carbon atoms are lost as CO2 in one revolution 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 and because succinate dehydrogenase does not differentiate between its two carboxyl groups, "randomization" of label occurs at this step such that all four carbon atoms of oxaloacetate appear to be labeled after one turn of the cycle. During gluconeogene-sis, some of the label in oxaloacetate is incorporated into glucose and glycogen (Figure 19-1). For a discussion of the stereochemical aspects of the citric acid cycle, see Greville (1968). The sites of inhibition (Q) by fluoroacetate, malonate, and arsenite are indicated.

When ketone bodies are being metabolized in extra-hepatic tissues there is an alternative reaction catalyzed by succinyl-CoA-acetoacetate-CoA transferase (thio-phorase)—involving transfer of CoA from succinyl-CoA to acetoacetate, forming acetoacetyl-CoA (Chapter 22).

The onward metabolism of succinate, leading to the regeneration of oxaloacetate, is the same sequence of chemical reactions as occurs in the P-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 fu-marate, is catalyzed by succinate dehydrogenase, which is bound to the inner surface of the inner mitochondrial membrane. The enzyme contains FAD and iron-sulfur (Fe:S) protein and directly reduces ubiquinone in the respiratory chain. Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate. Malate is converted to ox-aloacetate by malate dehydrogenase, a reaction requiring NAD+. Although the equilibrium of this reaction strongly favors malate, the net flux is toward the direction of oxaloacetate because of the continual removal of oxaloacetate (either to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to as-partate) and also because of the continual reoxidation of NADH.

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