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Home , Catabolism, Citric acid cycle, Fatty acid synthesis, Gluconeogenesis, Lipogenesis

ch16.qxd 2/13/2003 2:58 PM Page 130

The Cycle: The of Acetyl-CoA 16

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

BIOMEDICAL IMPORTANCE cated in the , either free or at- tached to the inner mitochondrial membrane, where The (Krebs cycle, tricarboxylic acid the of the respiratory chain are also found. cycle) is a series of reactions in mitochondria that oxi- dize acetyl residues (as acetyl-CoA) and reduce coen- zymes that upon reoxidation are linked to the forma- REACTIONS OF THE CITRIC ACID tion of ATP. CYCLE LIBERATE REDUCING The citric acid cycle is the final common pathway EQUIVALENTS & CO2 for the aerobic oxidation of , , and (Figure 16–3)* because , fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of The initial reaction between acetyl-CoA and oxaloac- the cycle. It also has a central role in , etate to form citrate is catalyzed by citrate , and interconversion of amino acids. Many which forms a -carbon bond between the methyl of these processes occur in most tissues, but the is carbon of acetyl-CoA and the carbonyl carbon of ox- the only in which all occur to a significant extent. aloacetate. The thioester bond of the resultant citryl- The repercussions are therefore profound when, for ex- CoA is hydrolyzed, releasing citrate and CoASH—an ample, large numbers of hepatic cells are damaged as in exergonic reaction. acute hepatitis or replaced by connective tissue (as in Citrate is isomerized to isocitrate by the cirrhosis). Very few, if any, genetic abnormalities of (aconitate hydratase); the reaction occurs in citric acid cycle enzymes have been reported; such ab- two steps: dehydration to cis-aconitate, some of which normalities would be incompatible with or normal remains bound to the enzyme; and rehydration to isoci- development. trate. Although citrate is a symmetric , aconi- tase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of THE CITRIC ACID CYCLE PROVIDES the cycle are not those that were added from acetyl- SUBSTRATE FOR THE CoA. This asymmetric behavior is due to channeling— RESPIRATORY CHAIN transfer of the product of directly onto the active site of aconitase without entering free solu- The cycle starts with reaction between the acetyl moiety tion. This provides integration of citric acid cycle activ- of acetyl-CoA and the four-carbon dicarboxylic acid ox- ity and the provision of citrate in the as a source aloacetate, forming a six-carbon tricarboxylic acid, cit- of acetyl-CoA for synthesis. The poison fluo- rate. In the subsequent reactions, two of CO2 roacetate is toxic because fluoroacetyl-CoA condenses are released and oxaloacetate is regenerated (Figure with oxaloacetate to form fluorocitrate, which inhibits 16–1). Only a small quantity of oxaloacetate is needed aconitase, causing citrate to accumulate. for the oxidation of a large quantity of acetyl-CoA; ox- Isocitrate undergoes dehydrogenation catalyzed by aloacetate may be considered to play a catalytic role. isocitrate to form, initially, oxalosucci- The citric acid cycle is an integral part of the process nate, which remains enzyme-bound and undergoes de- by which much of the free liberated during the to α-ketoglutarate. 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 forma- *From Circular No. 200 of the Committee of Editors of Biochemi- cal Journals Recommendations (1975): “According to standard tion of ATP (oxidative ; see Figure biochemical convention, the ending ate in, eg, palmitate, denotes 16–2 and also Chapter 12). This process is aerobic, re- any mixture of free acid and the ionized form(s) (according to pH) quiring oxygen as the final oxidant of the reduced in which the cations are not specified.” The same convention is coenzymes. The enzymes of the citric acid cycle are lo- adopted in this text for all carboxylic acids. 130 ch16.qxd 2/13/2003 2:58 PM Page 131

THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA / 131

Acetyl-CoA Carbohydrate Protein (C2)

CoA

Acetyl-CoA (C2)

H O Citrate Oxaloacetate Citrate Oxaloacetate 2 (C6) (C4) (C6) (C4) H2O Citric acid cycle Cis-aconitate (C6) H O Malate 2 (C4) 2H Isocitrate CO H2O 2 CO2 (C6)

2H CO2 Figure 16–1. Citric acid cycle, illustrating the cat- Fumarate (C4) α-Ketoglutarate alytic role of oxaloacetate. (C5) 2H NAD CO Succinate 2 (C ) Succinyl-CoA 2+ 2+ 4 requires Mg or Mn ions. There are three isoenzymes (C4) + P F of . One, which uses NAD , is 2H p + H O P found only in mitochondria. The other two use NADP 2 and are found in mitochondria and the cytosol. Respi- ratory chain-linked oxidation of isocitrate proceeds al- Q most completely through the NAD+-dependent en- zyme. Cyt b P Oxidative α-Ketoglutarate undergoes oxidative decarboxyla- phosphorylation tion in a reaction catalyzed by a multi-enzyme complex similar to that involved in the oxidative decarboxylation Cyt c of pyruvate (Figure 17–5). The -ketoglutarate dehy- drogenase complex requires the same cofactors as the complex—thiamin diphos- Cyt aa3 P 1/2 O phate, lipoate, NAD+, FAD, and CoA—and results in 2 the formation of succinyl-CoA. The equilibrium of this – Anaerobiosis reaction is so much in favor of succinyl-CoA formation (hypoxia, anoxia) H O that it must be considered physiologically unidirec- Respiratory chain 2 tional. As in the case of pyruvate oxidation (Chapter F 17), arsenite inhibits the reaction, causing the substrate, p Flavoprotein -ketoglutarate, to accumulate. Cyt Succinyl-CoA is converted to succinate by the en- zyme succinate thiokinase (succinyl-CoA synthe- P High-energy tase). This is the only example in the citric acid cycle of Figure 16–2. The citric acid cycle: the major catabo- substrate-level phosphorylation. Tissues in which glu- lic pathway for acetyl-CoA in aerobic organisms. Acetyl- coneogenesis occurs (the liver and ) contain two CoA, the product of carbohydrate, protein, and lipid ca- isoenzymes of succinate thiokinase, one specific for tabolism, is taken into the cycle, together with H O, and GDP and the other for ADP. The GTP formed is 2 used for the decarboxylation of oxaloacetate to phos- oxidized to CO2 with the release of reducing equivalents phoenolpyruvate in gluconeogenesis and provides a (2H). Subsequent oxidation of 2H in the respiratory chain leads to coupled phosphorylation of ADP to ATP. regulatory link between citric acid cycle activity and the withdrawal of oxaloacetate for gluconeogenesis. For one turn of the cycle, 11~ P are generated via ox- Nongluconeogenic tissues have only the isoenzyme that idative phosphorylation and one ~P arises at substrate uses ADP. level from the conversion of succinyl-CoA to succinate. ch16.qxd 2/13/2003 2:58 PM Page 132

CH3 CO* S CoA Acetyl-CoA O MALATE CITRATE SYNTHASE – DEHYDROGENASE C COO CoA SH – CH COO* – CH2 COO 2 NADH + H+ Oxaloacetate H O 2 HO C COO– HOCH COO* – NAD+ – CH2 COO – CH2 COO* Citrate L-Malate ACONITASE

FUMARASE Fe2+

H2O – Fluoroacetate CH2 COO* H2O C COO– H C COO* – CH COO– –OOC* C H Cis-aconitate Fumarate

FADH 2 H2O + SUCCINATE ACONITASE Fe2 DEHYDROGENASE FAD Malonate – CH2 COO* CH COO* – 2 CH COO– CH COO* – 2 HO CH COO– Succinate Isocitrate ATP NAD+

Mg2+ CoA SH NADH + H+ ADP + P i ISOCITRATE SUCCINATE – DEHYDROGENASE THIOKINASE CH2 COO* – CH2 COO* CH2 Arsenite NADH + H+ CH COO– O C S CoA + CO2 NAD – Succinyl-CoA – O C COO CH2 COO* α-KETOGLUTARATE Oxalosuccinate DEHYDROGENASE COMPLEX CH 2 Mn2+ ISOCITRATE CoA SH DEHYDROGENASE O C COO– CO2 α-Ketoglutarate

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 as- • terisk) and on the methyl carbon (using the designation ). Although two carbon atoms are lost as CO2 in one revo- lution 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 dehydro- genase 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 (Figure 19–1). For a discussion of the stereochemical aspects of the citric acid cycle, see Greville (1968). The sites of inhibition ( − ) by fluoroacetate, malonate, and arsenite are indicated.

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THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA / 133

When bodies are being metabolized in extra- the coenzyme for three in the cycle— hepatic tissues there is an alternative reaction catalyzed isocitrate dehydrogenase, α-ketoglutarate dehydrogen- by succinyl-CoA–acetoacetate-CoA transferase (thio- ase, and ; (3) thiamin ( phorase)—involving transfer of CoA from succinyl- B1), as thiamin diphosphate, the coenzyme for decar- CoA to acetoacetate, forming acetoacetyl-CoA (Chap- boxylation in the α-ketoglutarate dehydrogenase reac- ter 22). tion; and (4) , as part of , The onward of succinate, leading to the the attached to “active” resi- regeneration of oxaloacetate, is the same sequence of dues such as acetyl-CoA and succinyl-CoA. chemical reactions as occurs in the β-oxidation of fatty acids: dehydrogenation to form a carbon-carbon double bond, addition of to form a hydroxyl group, and THE CITRIC ACID CYCLE PLAYS A a further dehydrogenation to yield the oxo- group of PIVOTAL ROLE IN METABOLISM oxaloacetate. The first dehydrogenation reaction, forming fu- The citric acid cycle is not only a pathway for oxidation marate, is catalyzed by , which of two-carbon units—it is also a major pathway for in- is bound to the inner surface of the inner mitochondrial terconversion of arising from transamina- membrane. The enzyme contains FAD and iron-sulfur tion and of amino acids. It also provides (Fe:S) protein and directly reduces ubiquinone in the the substrates for synthesis by transamina- respiratory chain. (fumarate hydratase) cat- tion, as well as for gluconeogenesis and fatty acid syn- alyzes the addition of water across the double bond of thesis. Because it functions in both oxidative and syn- fumarate, yielding malate. Malate is converted to ox- thetic processes, it is (Figure 16–4). aloacetate by malate dehydrogenase, a reaction requir- + ing NAD . Although the equilibrium of this reaction The Citric Acid Cycle Takes Part in strongly favors malate, the net flux is toward the direc- Gluconeogenesis, , tion of oxaloacetate because of the continual removal of & Deamination oxaloacetate (either to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to as- All the intermediates of the cycle are potentially gluco- partate) and also because of the continual reoxidation genic, since they can give rise to oxaloacetate and thus of NADH. net production of glucose (in the liver and kidney, the organs that carry out gluconeogenesis; see Chapter 19). The key enzyme that catalyzes net transfer out of the TWELVE ATP ARE FORMED PER TURN cycle into gluconeogenesis is phosphoenolpyruvate OF THE CITRIC ACID CYCLE carboxykinase, which decarboxylates oxaloacetate to phosphoenolpyruvate, with GTP acting as the donor As a result of oxidations catalyzed by the dehydrogen- phosphate (Figure 16–4). ases of the citric acid cycle, three molecules of NADH Net transfer into the cycle occurs as a result of sev- and one of FADH2 are produced for each molecule of eral different reactions. Among the most important of acetyl-CoA catabolized in one turn of the cycle. These such is the formation of oxaloac- reducing equivalents are transferred to the respiratory etate by the carboxylation of pyruvate, catalyzed by chain (Figure 16–2), where reoxidation of each NADH . This reaction is important in results in formation of 3 ATP and reoxidation of maintaining an adequate concentration of oxaloacetate FADH2 in formation of 2 ATP. In addition, 1 ATP for the condensation reaction with acetyl-CoA. If acetyl- (or GTP) is formed by substrate-level phosphorylation CoA accumulates, it acts both as an allosteric activator catalyzed by succinate thiokinase. of pyruvate carboxylase and as an inhibitor of pyruvate dehydrogenase, thereby ensuring a supply of oxaloac- etate. Lactate, an important substrate for gluconeogene- PLAY KEY ROLES sis, enters the cycle via oxidation to pyruvate and then IN THE CITRIC ACID CYCLE carboxylation to oxaloacetate. Aminotransferase () reactions form Four of the are essential in the citric acid pyruvate from , oxaloacetate from aspartate, and cycle and therefore in energy-yielding metabolism: (1) α-ketoglutarate from glutamate. Because these reac- riboflavin, in the form of flavin adenine dinucleotide tions are reversible, the cycle also serves as a source of (FAD), a cofactor in the α-ketoglutarate dehydrogenase carbon skeletons for the synthesis of these amino acids. complex and in succinate dehydrogenase; (2) , in Other amino acids contribute to gluconeogenesis be- the form of adenine dinucleotide (NAD), cause their carbon skeletons give rise to citric acid cycle ch16.qxd 2/13/2003 2:58 PM Page 134

134 / CHAPTER 16

Hydroxyproline Lactate Cysteine Threonine Glycine TRANSAMINASE Alanine Pyruvate Acetyl-CoA PYRUVATE CARBOXYLASE PHOSPHOENOLPYRUVATE CARBOXYKINASE Phosphoenol- Glucose pyruvate Oxaloacetate

TRANSAMINASE Fumarate

Aspartate

Citrate

Isoleucine Methionine Succinyl-CoA CO2

α-Ketoglutarate Propionate

CO2 TRANSAMINASE Glutamate Figure 16–4. Involvement of the citric acid cycle in transamination and gluconeo- genesis. The bold arrows indicate the main pathway of gluconeogenesis.

intermediates. Alanine, cysteine, glycine, hydroxypro- Pyruvate dehydrogenase is a mitochondrial enzyme, line, serine, threonine, and tryptophan yield pyruvate; and is a cytosolic pathway, but the arginine, histidine, glutamine, and proline yield α-ke- mitochondrial membrane is impermeable to acetyl- toglutarate; , methionine, and valine yield CoA. Acetyl-CoA is made available in the cytosol from succinyl-CoA; and tyrosine and phenylalanine yield fu- citrate synthesized in the , transported marate (Figure 16–4). into the cytosol and cleaved in a reaction catalyzed by In , whose main metabolic fuel is short- ATP-citrate lyase. chain fatty acids formed by bacterial , the conversion of propionate, the major glucogenic product of fermentation, to succinyl-CoA via the Regulation of the Citric Acid Cycle methylmalonyl-CoA pathway (Figure 19–2) is espe- Depends Primarily on a Supply cially important. of Oxidized Cofactors The Citric Acid Cycle Takes Part In most tissues, where the primary role of the citric acid in Fatty Acid Synthesis cycle is in energy-yielding metabolism, respiratory (Figure 16–5) control via the respiratory chain and oxidative phos- phorylation regulates citric acid cycle activity (Chap- Acetyl-CoA, formed from pyruvate by the action of ter 14). Thus, activity is immediately dependent on the pyruvate dehydrogenase, is the major building block for supply of NAD+, which in turn, because of the tight long-chain fatty acid synthesis in nonruminants. (In ru- coupling between oxidation and phosphorylation, is de- minants, acetyl-CoA is derived directly from .) pendent on the availability of ADP and hence, ulti- ch16.qxd 2/13/2003 2:58 PM Page 135

THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA / 135

Fatty regulated in the same way as is pyruvate dehydrogenase Pyruvate Glucose acids (Figure 17–6). Succinate dehydrogenase is inhibited by oxaloacetate, and the availability of oxaloacetate, as controlled by malate dehydrogenase, depends on the + [NADH]/[NAD ] ratio. Since the Km for oxaloacetate PYRUVATE of citrate synthase is of the same order of magnitude as DEHYDROGENASE Acetyl-CoA the intramitochondrial concentration, it is likely that Acetyl-CoA the concentration of oxaloacetate controls the rate of citrate formation. Which of these mechanisms are im- Oxaloacetate portant in vivo has still to be resolved. Citric ATP-CITRATE acid LYASE cycle SUMMARY Oxaloacetate Citrate Citrate • The citric acid cycle is the final pathway for the oxi- dation of carbohydrate, lipid, and protein whose common end-, acetyl-CoA, reacts with ox- aloacetate to form citrate. By a series of dehydrogena- tions and , citrate is degraded, CO CO releasing reduced coenzymes and 2CO2 and regener- 2 2 ating oxaloacetate. MITOCHONDRIAL • The reduced coenzymes are oxidized by the respira- MEMBRANE tory chain linked to formation of ATP. Thus, the Figure 16–5. Participation of the citric acid cycle in cycle is the major route for the generation of ATP fatty acid synthesis from glucose. See also Figure 21–5. 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 mately, on the rate of utilization of ATP in chemical to oxidation it is important in the provision of car- and physical work. In addition, individual enzymes of bon skeletons for gluconeogenesis, fatty acid synthe- the cycle are regulated. The most likely sites for regula- sis, and interconversion of amino acids. tion are the nonequilibrium reactions catalyzed by pyruvate dehydrogenase, citrate synthase, isocitrate de- REFERENCES hydrogenase, and α-ketoglutarate dehydrogenase. The dehydrogenases are activated by Ca2+, which increases Baldwin JE, Krebs HA: The evolution of metabolic cycles. Nature in concentration during muscular contraction and se- 1981;291:381. cretion, when there is increased energy demand. In a Goodwin TW (editor): The Metabolic Roles of Citrate. Academic tissue such as brain, which is largely dependent on car- Press, 1968. bohydrate to supply acetyl-CoA, control of the citric Greville GD: Vol 1, p 297, in: and Its Disorders. Dickens F, Randle PJ, Whelan WJ (editors). Acad- acid cycle may occur at pyruvate dehydrogenase. Sev- emic Press, 1968. eral enzymes are responsive to the energy status, as + Kay J, Weitzman PDJ (editors): Krebs’ Citric Acid Cycle—Half a shown by the [ATP]/[ADP] and [NADH]/[NAD ] ra- Century and Still Turning. Biochemical Society, London, tios. Thus, there is allosteric inhibition of citrate syn- 1987. thase by ATP and long-chain fatty acyl-CoA. Allosteric Srere PA: The enzymology of the formation and breakdown of cit- activation of mitochondrial NAD-dependent isocitrate rate. Adv Enzymol 1975;43:57. dehydrogenase by ADP is counteracted by ATP and Tyler DD: The Mitochondrion in Health and Disease. VCH Pub- NADH. The α-ketoglutarate dehydrogenase complex is lishers, 1992.