Harper's Biochemistry Chapter 16 - The Citric Acid Cycle.PDF

C H A P T E R The Citric Acid Cycle: A Pathway Central to Carbohydrate, Lipid, & 16 Amino Acid Metabolism Owen P. McGuinness, PhD OBJ E C TI VE S Describe...

C H A P T E R The Citric Acid Cycle: A Pathway Central to Carbohydrate, Lipid, & 16 Amino Acid Metabolism Owen P. McGuinness, PhD OBJ E C TI VE S Describe the reactions of the citric acid cycle. Identify the reactions that generate reducing equivalents that are oxidized in After studying this chapter, the mitochondrial electron transport chain to yield ATP. you should be able to: Identify the steps that require vitamins in the citric acid cycle. Explain how the citric acid cycle provides both a route for catabolism of amino acids and a route for their synthesis. Describe the main anaplerotic and cataplerotic pathways that permit replenishment and removal of citric acid cycle intermediates. 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 impair citric acid cycle flux. BIOMEDICAL IMPORTANCE are associated with severe neurologic damage as a result of impaired ATP formation in the central nervous system. The citric acid cycle (the Krebs or tricarboxylic acid cycle) is a Hyperammonemia, as occurs in advanced liver disease, sequence of reactions in mitochondria that oxidizes the acetyl leads to loss of consciousness, coma, and convulsions. The moiety of acetyl-CoA to CO2 and couples this to the reduc- impaired activity of the citric acid cycle leads to reduced for- tion of coenzymes that are reoxidized in the electron transport mation of ATP. Ammonia both depletes citric acid cycle inter- chain (see Chapter 13), linked to the formation of ATP. mediates (by withdrawing α-ketoglutarate for the formation of The citric acid cycle is the final common pathway for the glutamate and glutamine) and inhibits the oxidative decarbox- oxidation of carbohydrate, lipid, and protein because glucose, ylation of α-ketoglutarate. 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 THE CITRIC ACID CYCLE is the only tissue in which all occur to a significant extent. The PROVIDES SUBSTRATES repercussions are therefore profound when, for example, large FOR THE RESPIRATORY CHAIN numbers of hepatic cells are damaged as in acute hepatitis or The cycle starts with reaction between the acetyl moiety of replaced by connective tissue (as in cirrhosis). The few genetic acetyl-CoA and the four-carbon dicarboxylic acid oxaloac- defects of citric acid cycle enzymes that have been reported etate, forming a six-carbon tricarboxylic acid, citrate. In the subsequent reactions, two molecules of CO2 are released This was adapted from chapter in 30th edition by David A. Bender, and oxaloacetate is regenerated (Figure 16–1). Only a small PhD, & Peter A. Mayes, PhD, DSc quantity of oxaloacetate is needed for the oxidation of a large 156  CHAPTER 16 The Citric Acid Cycle: A Pathway Central to Carbohydrate, Lipid, & Amino Acid Metabolism 157 Acetyl-CoA (C2) CoA Oxaloacetate Citrate (C4) (C6) CO2 CO2 FIGURE 16–1 The citric acid cycle, illustrating the impor- tance of regenerating oxaloacetate to sustain the cycle. quantity of acetyl-CoA; it can be considered as playing a catalytic role, since it is regenerated at the end of the cycle. The citric acid cycle provides the main pathway for ATP formation linked to the oxidation of metabolic fuels. During the oxidation of acetyl-CoA, coenzymes are reduced, then reoxidized in the respiratory chain, linked to the formation of ATP (oxidative phosphorylation, Figure 16–2; 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. This is where the enzymes and coen- zymes of the respiratory chain are also found (see Chapter 13). To sustain the cycle, the number of carbons entering and leaving must be equal. A two-carbon molecule (Acetyl-CoA) FIGURE 16–2 The citric acid cycle: the major catabolic combines with a four-carbon molecule (oxaloacetate) to form a pathway for acetyl-CoA. Acetyl-CoA, the product of carbohydrate, protein, and lipid catabolism, enters the cycle by forming citrate, and six-carbon molecule (citrate). After one turn of the cycle citrate is oxidized to CO2 with the reduction of coenzymes. Reoxidation of is converted back to oxaloacetate and two carbons are released the coenzymes in the respiratory chain leads to phosphorylation of as CO2. If a metabolic pathway adds carbon to the cycle that is ADP to ATP. For one turn of the cycle, nine ATP are generated via oxi- not though acetyl-CoA (pyruvate [C3] to oxaloacetate [C4] for dative phosphorylation and one ATP (or GTP) arises at substrate level gluconeogenesis; see Chapter 19) then there must be an outlet from the conversion of succinyl-CoA to succinate. pathway to bring an equal amount of carbon out (oxaloacetate [C4] to phosphoenolpyruvate [C3]) of the cycle. The entry of (Figure 16–3). The thioester bond of the resultant citryl-CoA carbon is called anaplerosis. The exiting of carbon is called is hydrolyzed, releasing citrate and CoASH—an exothermic cataplerosis. Thus to sustain the citric acid cycle anaplerosis reaction. The coenzyme A released can be recycled in the con- must equal cataplerosis. version of pyruvate to acetyl-CoA by the pyruvate dehydroge- nase complex. Citrate is isomerized to isocitrate by the enzyme aconi- REACTIONS OF THE CITRIC ACID tase (aconitate hydratase); the reaction occurs in two steps: CYCLE GENERATE REDUCING dehydration to cis-aconitate and rehydration to isocitrate. Although citrate is a symmetrical molecule, aconitase reacts EQUIVALENTS & CO2 with citrate asymmetrically, so that the two carbon atoms that The initial reaction between acetyl-CoA and oxaloacetate are lost in subsequent reactions of the cycle are not those that (C4) to form citrate (C6) is catalyzed by citrate synthase, were added from acetyl-CoA. This asymmetric behavior is which forms a carbon–carbon bond between the methyl car- the result of channeling—transfer of the product of citrate bon of acetyl-CoA and the carbonyl carbon of oxaloacetate synthase directly onto the active site of aconitase, without 158 SECTION IV Metabolism of Carbohydrates FIGURE 16–3 The citric acid (Krebs) cycle. Oxidation of NADH and FADH2 in the respiratory chain leads to the formation of ATP via oxi- dative phosphorylation. In order to follow the passage of acetyl-CoA through the cycle, the two carbon atoms of the acetyl moiety 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 symmetrical 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 (see Figure 20–1). The sites of inhibition (⊝) by fluoroacetate, malonate, and arsenite are indicated. entering free solution. As fatty acid synthesis uses an anaple- of the cycle (cataplerosis) to be a source of acetyl-CoA for fatty rotic pathway (pyruvate carboxylase) to add oxaloacetate to the acid synthesis. This allows citric acid cycle activity to be main- cycle, this in turn will make extra citrate and thus isocitrate. tained to generate ATP and reducing equivalents. This energy The excess isocitrate will act as a break on aconitase. Because will be needed to support the energy demanding process of of channeling, citrate is only available in free solution to be fatty acid synthesis while providing citrate in the cytosol as a transported from the mitochondria to the cytosol for fatty acid source of acetyl-CoA for fatty acid synthesis. synthesis when aconitase is inhibited by accumulation of its The poison fluoroacetate is found in some of the plants, product, isocitrate. The “free” citrate can then be exported out and their consumption can be fatal to grazing animals. Some  CHAPTER 16 The Citric Acid Cycle: A Pathway Central to Carbohydrate, Lipid, & Amino Acid Metabolism 159 fluorinated compounds used as anticancer agents and indus- the inner surface of the inner mitochondrial membrane. The trial chemicals (including pesticides) are metabolized to fluoro- enzyme contains FAD and iron-sulfur (Fe-S) protein, and acetate. It is toxic because fluoroacetyl-CoA condenses with directly reduces ubiquinone in the electron transport chain. oxaloacetate to form fluorocitrate, which inhibits aconitase, Fumarase (fumarate hydratase) catalyzes the addition of causing citrate to accumulate. water across the double bond of fumarate, yielding malate. Isocitrate undergoes dehydrogenation catalyzed by isoci- Malate is oxidized to oxaloacetate by malate dehydrogenase, trate dehydrogenase to form, initially, oxalosuccinate, which linked to the reduction of NAD+ to form NADH. Although the remains enzyme bound and undergoes decarboxylation to equilibrium of this reaction strongly favors malate, the net flux α-ketoglutarate. The decarboxylation requires Mg2+ or Mn2+ is to oxaloacetate because oxaloacetate is rapidly being used. ions. There are three isoenzymes of isocitrate dehydrogenase. Oxaloacetate is used for multiple reactions (form citrate, leave One, which uses nicotinamide adenine dinucleotide (NAD+), the mitochondria to be a substrate for gluconeogenesis, or to is found only in mitochondria. The other two use NADP+ and undergo transamination to form aspartate). NADH is reoxi- are found in mitochondria and the cytosol. Respiratory chain– dized to NAD by the respiratory chain. linked oxidation of isocitrate occurs through the NAD+- dependent enzyme. α-Ketoglutarate undergoes oxidative decarboxylation TEN ATP ARE FORMED PER TURN in a reaction catalyzed by a multienzyme complex similar to OF THE CITRIC ACID CYCLE that involved in the oxidative decarboxylation of pyruvate (see Figure 17–5). The α-ketoglutarate dehydrogenase complex As a result of oxidations catalyzed by the dehydrogenases of requires the same cofactors as the pyruvate dehydrogenase the citric acid cycle, three molecules of NADH and one of complex—thiamin diphosphate, lipoate, NAD+, flavin adenine FADH2 are produced for each molecule of acetyl-CoA catabo- dinucleotide (FAD), and CoA—and results in the formation lized in one turn of the cycle. These reducing equivalents are of succinyl-CoA. The equilibrium of this reaction is so much transferred to the respiratory chain (see Figure 13–3), where in favor of succinyl-CoA formation that it must be considered reoxidation of each NADH results in formation of ~2.5 ATP, to be physiologically unidirectional. As in the case of pyru- and of FADH2, ~1.5 ATP. In addition, 1 ATP (or GTP) is vate oxidation (see Chapter 17), arsenite inhibits the reac- formed by substrate-level phosphorylation catalyzed by suc- tion, causing the substrate, α-ketoglutarate, to accumulate. cinate thiokinase. High concentrations of ammonia seen in liver disease inhibits α-ketoglutarate dehydrogenase. Succinyl-CoA is converted to succinate by the enzyme VITAMINS PLAY KEY ROLES succinate thiokinase (succinyl-CoA synthetase). This is the IN THE CITRIC ACID CYCLE only example of substrate-level phosphorylation (transfer of a Four of the B vitamins (see Chapter 44) are essential in the phosphate group bound to the enzyme to GDP or ADP with citric acid cycle and hence energy-yielding metabolism: ribo- generation of ATP or GTP) in the citric acid cycle. Tissues in flavin, as FAD, is the cofactor for succinate dehydrogenase; which gluconeogenesis occurs (the liver and kidney) contain niacin, as NAD+, is the electron acceptor for isocitrate dehy- two isoenzymes of succinate thiokinase, one specific for GDP drogenase, α-ketoglutarate dehydrogenase, and malate dehy- and the other for ADP. The GTP formed is used for the decar- drogenase; thiamin (vitamin B1), as thiamin diphosphate, boxylation of oxaloacetate to phosphoenolpyruvate in gluco- is the coenzyme for decarboxylation in the α-ketoglutarate neogenesis, and provides a regulatory link between citric acid dehydrogenase reaction; and pantothenic acid, as part of cycle activity and the withdrawal (cataplerosis) of oxaloacetate coenzyme A, is esterified to carboxylic acids to form acetyl- for gluconeogenesis. Nongluconeogenic tissues have only the CoA and succinyl-CoA. isoenzyme that phosphorylates ADP. When ketone bodies are being metabolized in extrahepatic tissues, there is an alternative reaction catalyzed by succinyl- THE CITRIC ACID CYCLE PLAYS CoA–acetoacetate-CoA transferase (thiophorase), involving transfer of CoA from succinyl-CoA to acetoacetate, forming A PIVOTAL ROLE IN METABOLISM acetoacetyl-CoA and succinate (see Chapter 22). The citric acid cycle is not only a pathway for oxidation of two The onward metabolism of succinate, leading to the carbon units, but is also a major pathway for interconversion regeneration of oxaloacetate, is the same sequence of chemical of metabolites arising from transamination and deamination reactions as occurs in the β-oxidation of fatty acids: dehydro- of amino acids (see Chapters 28 and 29), and providing the genation to form a carbon–carbon double bond, addition of substrates for amino acid synthesis by transamination (see water to form a hydroxyl group, and a further dehydrogena- Chapter 27), as well as for gluconeogenesis (see Chapter 19) tion to yield the oxo-group of oxaloacetate. and fatty acid synthesis (see Chapter 23). Because it functions The first dehydrogenation reaction, forming fumarate, in both oxidative and synthetic processes, it is amphibolic is catalyzed by succinate dehydrogenase, which is bound to (Figure 16–4). 160 SECTION IV Metabolism of Carbohydrates FIGURE 16–4 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 and an inhibitor of pyruvate dehydrogenase, thereby ensur- ing a supply of oxaloacetate. Lactate, an important substrate in Gluconeogenesis, Transamination, for gluconeogenesis, enters the cycle via oxidation to pyru- & Deamination vate and then carboxylation to oxaloacetate. Glutamate and All the intermediates of the cycle are potentially glucogenic, glutamine are important anaplerotic substrates. They yield since they can give rise to oxaloacetate, and hence produc- α-ketoglutarate as a result of the reactions catalyzed by glu- tion of glucose (in the liver and kidney, which carry out glu- taminase and glutamate dehydrogenase. Transamination of coneogenesis; see Chapter 19). The key enzyme that catalyzes aspartate leads directly to the formation of oxaloacetate, and transfer out of the cycle into gluconeogenesis is phosphoenol- a variety of compounds that are metabolized can yield propio- pyruvate carboxykinase, which catalyzes the decarboxylation nyl CoA, which can be carboxylated and isomerized to suc- of oxaloacetate to phosphoenolpyruvate, with GTP acting as cinyl CoA, are also important anaplerotic substrates. the phosphate donor (see Figure 19–1). The GTP required for Aminotransferase (transaminase) reactions form pyruvate this reaction is provided by the GDP-dependent isoenzyme of from alanine, oxaloacetate from aspartate, and α-ketoglutarate succinate thiokinase. This ensures that oxaloacetate will not be from glutamate. Because these reactions are reversible, the withdrawn from the cycle for gluconeogenesis unless GTP was cycle also serves as a source of carbon skeletons (ie, cataplero- supplied. Otherwise this would lead to depletion of citric acid sis) for the synthesis of these amino acids. Other amino acids cycle intermediates, and hence reduced generation of ATP. contribute to gluconeogenesis because their carbon skeletons Net transfer into the cycle (ie, anaplerosis) occurs as a give rise to citric acid cycle intermediates. Alanine, cysteine, result of several reactions. Among the most important of such glycine, hydroxyproline, serine, threonine, and tryptophan anaplerotic reactions is the formation of oxaloacetate by the yield pyruvate; arginine, histidine, glutamine, and proline carboxylation of pyruvate, catalyzed by pyruvate carboxylase yield α-ketoglutarate; isoleucine, methionine, and valine yield (see Figure 16–4). This reaction is important in maintaining succinyl-CoA; tyrosine and phenylalanine yield fumarate (see an adequate concentration of oxaloacetate for the condensa- Figure 16–4). tion reaction with acetyl-CoA. If acetyl-CoA accumulates, The citric acid cycle itself does not provide a pathway for it acts as both an allosteric activator of pyruvate carboxylase the complete oxidation of the carbon skeletons of amino acids  CHAPTER 16 The Citric Acid Cycle: A Pathway Central to Carbohydrate, Lipid, & Amino Acid Metabolism 161 that give rise to intermediates such as α-ketoglutarate, succi- The Citric Acid Cycle Takes Part in nyl CoA, fumarate, and oxaloacetate, because this results in an increase in the amount of oxaloacetate. For complete oxida- Fatty Acid Synthesis tion to occur a cataplerotic route has to be used. Oxaloacetate Acetyl-CoA, formed from pyruvate by the action of pyruvate must leave the cycle to undergo phosphorylation and carbox- dehydrogenase, is the major substrate for long-chain fatty acid ylation to phosphoenolpyruvate (at the expense of GTP), then synthesis in nonruminants (Figure 16–5). (In ruminants, be dephosphorylated to form pyruvate (catalyzed by pyruvate acetyl-CoA is derived directly from acetate.) Pyruvate dehy- kinase). Pyruvate can then undergo oxidative decarboxylation drogenase is a mitochondrial enzyme, and fatty acid synthesis to acetyl-CoA (catalyzed by pyruvate dehydrogenase). is in the cytosol; the mitochondrial membrane is impermeable In ruminants, main metabolic fuel is short-chain fatty to acetyl-CoA. For acetyl-CoA to be available in the cytosol, acids formed by bacterial fermentation and the formation of citrate is transported from the mitochondrion to the cyto- propionate. Propionate is the major glucogenic product of sol, then cleaved in a reaction catalyzed by citrate lyase (see rumen fermentation. It enters the cycle at succinyl-CoA via Figure 16–5). Citrate is only available for transport out of the the methylmalonyl-CoA pathway (see Figure 19–2) to form mitochondrion when aconitase is inhibited by its product and glucose via gluconeogenesis. therefore saturated with its substrate, so that citrate cannot be Glycolysis in cytosol CH3 C O COO– Pyruvate NAD+ Pyruvate dehydrogenase NADH CO2 CH3 C O SCoA CoASH – Acetyl CoA COO COO– COO– C O CH 2 CH 2 CH2 Citrate synthase HO C COO– HO C COO– – COO CH 2 CH 2 Oxaloacetate COO– COO– Citrate CoASH Citrate lyase ADP + Pi CO2 CH3 ATP CH3 C O C O SCoA Pyruvate carboxylase COO– COO– Acetyl CoA C O for fatty acid synthesis CH2 COO– Oxaloacetate NADH Malate dehydrogenase NAD+ CO2 COO – CH3 Malic enzyme HC OH C O CH2 COO– NADP+ COO– NADPH Pyruvate Malate FIGURE 16–5 Participation of the citric acid cycle in provision of cytosolic acetyl-CoA for fatty acid synthesis from glucose. See also Figure 23–5. 162 SECTION IV Metabolism of Carbohydrates channeled directly from citrate synthase onto aconitase. This Thus, there is allosteric inhibition of citrate synthase by ATP ensures that citrate is used for fatty acid synthesis only when and long-chain fatty acyl-CoA. Allosteric activation of mito- there is an adequate amount to ensure continued activity of the chondrial NAD-dependent isocitrate dehydrogenase by ADP cycle. The identity and regulation of the many mitochondrial is counteracted by ATP and NADH. The α-ketoglutarate dehy- transport systems (eg, malate, citrate, pyruvate) involved in drogenase complex is regulated in the same way as is pyruvate cataplerosis and anaplerosis pathways are poorly understood, dehydrogenase (see Figure 17–6). Succinate dehydrogenase is but they likely play a critical regulatory role in these pathways. inhibited by oxaloacetate, and the availability of oxaloacetate The oxaloacetate released by citrate lyase cannot reenter is controlled by malate dehydrogenase and depends on the the mitochondrion, but is reduced to malate, at the expense of [NADH]/[NAD+] ratio. Since the Km of citrate synthase for NADH, and the malate undergoes oxidative decarboxylation oxaloacetate is of the same order of magnitude as the intrami- to pyruvate, reducing NADP+ to NADPH. This reaction, cata- tochondrial concentration, it is likely that the concentration of lyzed by the malic enzyme, is the source of half the NADPH oxaloacetate controls the rate of citrate formation. required for fatty acid synthesis (the remainder is provided Hyperammonemia, as occurs in advanced liver disease and by the pentose phosphate pathway; see Chapter 20). Pyruvate a number of (rare) genetic diseases of amino acid metabolism, enters the mitochondrion and is carboxylated to oxaloacetate leads to loss of consciousness, coma and convulsions, and may by pyruvate carboxylase, an ATP-dependent reaction in which be fatal. This is largely because of the withdrawal (cataplerosis) the coenzyme is the vitamin biotin. of α-ketoglutarate to form glutamate (catalyzed by glutamate dehydrogenase) and then glutamine (catalyzed by glutamine Regulation of the Citric Acid Cycle synthetase) from the citric acid cycle, which is not matched by anaplerosis. It lowers concentrations of all citric acid cycle inter- Depends Primarily on a Supply mediates, citric acid cycle flux, and the generation of ATP. The of Oxidized Cofactors equilibrium of glutamate dehydrogenase is finely poised, and In most tissues, where the primary role of the citric acid cycle the direction of reaction depends on the ratio of NAD+:NADH is in energy-yielding metabolism, respiratory control via the and the concentration of ammonium ions, which is elevated respiratory chain and oxidative phosphorylation regulates citric in liver disease. In addition, ammonia inhibits α-ketoglutarate acid cycle activity (see Chapter 13). Thus, cycle activity is imme- dehydrogenase, and possibly also pyruvate dehydrogenase fur- diately dependent on the supply of NAD+, which in turn, because ther decreasing citric acid cycle flux. of the tight coupling between oxidation and phosphorylation, is dependent on the availability of ADP and hence, ultimately on the utilization of ATP in chemical and physical work. Thus as SUMMARY ATP usage increases (eg, muscle contraction) this will gener- The citric acid cycle is the final pathway for the oxidation ate ADP to help drive the cycle. If ATP demand is low the cycle of carbohydrate, lipid, and protein. Their common end- will slow down to match the demand. In addition, individual metabolite, acetyl-CoA, reacts with oxaloacetate to form citrate. By a series of dehydrogenations and decarboxylations, enzymes of the cycle are regulated. In general these regulatory citrate is degraded, reducing coenzymes, releasing two CO2, events help to couple rapid changes in energy demand with citric and regenerating oxaloacetate. acid cycle flux. The main sites for regulation are the nonequi- The reduced coenzymes are oxidized by the respiratory chain librium reactions catalyzed by pyruvate dehydrogenase, citrate linked to formation of ATP. Thus, the cycle is the major synthase, isocitrate dehydrogenase, and α-ketoglutarate dehy- pathway for the formation of ATP. It is located in the matrix of drogenase. The dehydrogenases are activated by Ca2+, which mitochondria adjacent to the enzymes of the respiratory chain increases in concentration during contraction of muscle and and oxidative phosphorylation. during secretion by other tissues, when there is increased energy The citric acid cycle is amphibolic, since in addition to demand. In a tissue such as brain, which is largely dependent on oxidation it is important in the provision of carbon skeletons glucose to supply acetyl-CoA, control of the citric acid cycle may for gluconeogenesis, acetyl-CoA for fatty acid synthesis, and occur at pyruvate dehydrogenase. interconversion of amino acids. For these processes to be Several enzymes are also responsive to the energy status sustained they rely on a balance of anaplerosis and cataplerosis as reflected by the [ATP]/[ADP] and [NADH]/[NAD+] ratios. in the citric acid cycle.