Chapter 10: The Citrate Cycle

The citrate cycle The first reaction of the citrate cycle is catalyzed by the enzyme citrate synthase. The product of this reaction is citrate, which is the conjugate base of citric acid Chemically, the distinctive feature of citric acid is its three carboxylic acid groups, which are ionized at physiologic pH The citrate cycle is not simply an extension of glycolysis. Rather, it initiates the production of energy from fatty acids and proteins (amino acids), as well as from carbohydrates (glucose), linking several metabolic pathways The citrate cycle is considered the "hub" of cellular metabolism for three primary reasons: 1. It is central to aerobic metabolism and ATP production by generating the bulk of NADH and FADH2, which are oxidized by the electron transport system to generate ATP by oxidative phosphorylation 2. It links the oxidation of various metabolic fuels (carbohydrates, fatty acids, and proteins) to ATP synthesis through shared intermediates 3. It provides metabolites for numerous biosynthetic pathways 10.1 The citrate cycle captures energy using redox reactions Overview of the citrate cycle In eukaryotic cells, all of the enzymes in the citrate cycle and electron transport system reside in the mitochondrial inner membrane and matrix Acetyl-CoA, moreover, is produced by faulty acid oxidation as a major energy source in most organisms In doing so, each turn of the citrate cycle intermediates to NAD+ and FAD, generating 3 NADH and 1 FADH2 The citrate cycle also contains one substrate-level phosphorylation reaction, which generates I GTP for every turn of the cycle. This GTP is used by the enzyme nucleosome diphosphate kinase to phosphorylate ADP to make ATP On the basis of this ATP currency exchange ratio and the one substrate-level phosphorylation reaction, each turn of the cycle produces ~10 ATP for every acetyl-CoA molecule that is oxidized Two features of the citrate cycle distinguish it from linear metabolic pathways such as glycolysis First, oxaloacetate is both the substrate for the first reaction (catalyzed by citrate synthase) and the product of the last reaction (catalyzed by malate dehydrogenase) The second important feature of the citrate cycle is that the enzymes pyruvate dehydrogenase and pyruvate carboxylase control the flow of acetyl-CoA and oxaloacetate, respectively, into the citrate cycle Redox reactions involve the loss and gain of electrons The energy available from redox reactions is due to differences in the electron affinity of two compounds and is an inherent property of each molecule based on molecular structure Each redox reaction consists of two half-reactions: (1) an oxidation reaction (a loss of electrons) and (2) a reduction reaction (a gain of electrons) Compounds that accept elections are called oxidants (or oxidizing agents) and are reduced in the reaction, whereas compounds that donate electrons are called reductants (or reducing agents) and are oxidized in the reaction Fe3+ + Cu+ e- Cu2+ Fe3+ + Cu+ Fe2+ (reduction of Fe2+) + e- (oxidation of Cu+) Fe2+ + Cu2+ Enzymes that catalyze biochemical redox reactions are strictly called oxidoreductases, but most oxidation reactions involve the loss of one or more hydrogen atoms, so these enzymes are often called dehydrogenases Free energy changes can be calculated from reduction potential differences The two primary energy-conversion reactions in metabolism are (1) phosphoric transfers involving ATP and (2) redox reactions that use the electron carriers NAD+/NADH and FAD/FADH2 The E of oxidants with a higher affinity for electrons than that of H+ are recorded as positive E values (E > 0). Oxidants with a lower affinity for elections than that of H+ are recorded as negative E values (E < 0) The amount of energy available from a coupled redox reaction is directly related to the difference between two reduction potentials, denoted as delta E From the definition of delta E , this means for a coupled redox reaction to be favorable, the reduction potential of the e- acceptor needs to be more positive than that of the e- donor In a spontaneous coupled redox reaction such as this, electrons flow from the reluctant in the conjugate redox pair with the lower E value (more negative) toward the oxidant in the conjugate redox pair with the higher E value (less negative) 10.2 Pyruvate dehydrogenase converts pyruvate to acetyl-CoA Pyruvate must be transported from the cytosol into the mitochondrial matrix before it can serve as a source of reducing power for the cell (used in the citrate cycle) or as a precursor for glucose synthesis (used in gluconeogenesis) When the energy charge in the cell is low, pyruvate is metabolized in the mitochondria by the multi-subunit enzyme complex pyruvate dehydrogenase Five coenzymes are required for the pyruvate dehydrogenase reaction Generally, enzymes use amino acid side chains to catalyze chemical reactions in the active site of the protein. Amino acids, though, have relatively limited chemistry that they can accomplish Therefore, enzyme cofactors and coenzymes are often involved in mediating enzyme catalysis, especially in redox reactions, where they function as electron carriers The five coenzymes required for the pyruvate dehydrogenase reaction are NAD+, FAD, CoA, thiamine pyrophosphate (TPP), and a-lipoic acid (lipoamide) Nicotinamide adenine dinucleotide (NAD+) is derived from the water-soluble vitamin niacin, which is also called nicotinic acid, or vitamin B3 NAD+, and its phosphorylated form NADP+, are involved in at least 200 redox reactions in the cell, each of which is characterized by the transfer of 2 e- in the form of a hydride ion (CH-) transfer Catabolic redox reactions primarily use the conjugate redox pair NAD+/NADH, whereas anabolic reactions use NADP+/NADPH Flavin adenine dinucleotide (FAD) is derived from the water-soluble vitamin riboflavin, which is also called NAD+ vitamin B2 FAD is a coenzyme in the pyruvate dehydrogenase n complex and is also covalently bound to a histidine residue in the citrate cycle enzyme succinate dehydrogenase mom Unlike NAD+, FAD can accept one electron at a time 0 and form a partially reduced intermediate called OHOH semiquinone N FADH2 functions as a reluctant in the pyruvate o O dehydrogenase complex by transferring two a msn.io electrons to NAD+ in the final step of the reaction to generate NADH+ + H+ Coenzyme A (CoA) is derived from the watersoluble vitamin pantothenic acid, which is also called OHOH vitamin B5 The transported acetate unit of acetyl-CoA is covalently attached to CoA through an activated this ester bond, which has a high standard free energy of MMM hydrolysis, making it an ideal acyl carrier compound ay Like many other coenzymes, CoA does not readily cross cell membranes. 0 MMM Mmm Therefore, CoA is degraded by enzymes in the gut to yield pantothenic acid, which is absorbed and O Riboflavin transported to tissues through the circulatory system Thiamine pyrophosphate (TPP) is derived from the water-soluble Hz vitamin thiamine, which is also called vitamin B1 Thiamine is absorbed in the gut and o 0 transported to tissues, where it is phosphorylated by the enzyme thiamine pyrophosphokinase in the O P O presence of ATP to form TPP and AMP o pp The role of a-lipoic acid in metabolic some mom omi reactions is to provide a reactive disulfide that can participate in redox reactions within the enzyme active OH OH site The pyruvate dehydrogenase complex is a metabolic machine The eukaryotic pyruvate dehydrogenase complex contains more than 90 subunits, which consist of three different catalytic enzymes: (1) E1, pyruvate dehydrogenase; (2) E2, dihydrolipoyl acetyltransferase; and (3) E3, dihydrolipoyl dehydrogenase Three of the coenzymes are covalently linked to enzyme subunits: TPP is attached to the E1 subunit (pyruvate dehydrogenase), lipoamide is the functional component of the E2 subunit (dihydrolipoyl acetyltransferase), and FAD is covalently bound to the E3 subunit (dihydrolipoyl dehydrogenase) t.fi regenerating oxidized form of lipoamide and formation of acetyl-CoA reducing NAD+ to NADH 1. The E1 subunit (pyruvate dehydrogenase) binds pyruvate and catalyzes a decarboxylation reaction, resulting in the formation of hydroxyethyl-TPP and the subsequent release of CO2 2. The hydroxyethyl-TPP moiety of E1 reacts with the disulfide of the lipoamide group on the N-terminal domain of the E2 subunit (dihydrolipoyl acetyltransferase). This generates acetyl-dihydrolipodmide through a thioester bond 3. The E2 acetyl-dihydrolipoamide group carries the acetal group from the E1 catalytic site across a gap in the complex to the E2 catalytic site, where it reacts with CoA to yield acetyl-CoA and fully reduced dihydrolipoamide 4. The dihydrolipoamide group then swings over to the E3 subunit (dihydrolipoyl dehydrogenase), where it is reoxidized to the disulfide by a transfer of 2 e- and 2 H+ to a disulfide contained on the E3 submit, forming a reduced dithiol. The reduced dithiol on the E3 subunit is reoxidized by transferring 2 e- and 2 H+ to the E3-linked FAD moiety, transiently forming E3-FADH2 5. The E3-FADH2 coenzyme intermediate is reoxidized in a coupled redox reaction that transfers 2 e- to NAD+, producing NADH + H+ TPP pyruvate acetyl-dihydrolipoamide N ya O r S O its CoA R 0 0 3 CoA acetyl-CoA 2 El Is 3FAD CO dihydrolipoamide s MS y pp OH hydroxyethyl-TPP FADH2 MM R lipoamide as 5 NADH + H+ NAD+ This E2 amino-terminal bridge consists of four functional components: (1) the ball, the lipoamide moiety covalently linked to the 84-amino-acid E2 lipoyl domain (2) the chain, the 62-amino-acid flexible segment of E2 (3) the pivot, the 35-amino-acid E1/E3 binding domain (4) the linker, the 40-amino-acid segment needed to reorient the pivot between the E1 and E3 subunits Because of the relative positioning of the E2 and E1 subunits within the pyruvate dehydrogenase complex, a single E2 subunit can obtain hydroxyethyl groups from multiple E1 catalytic sites and deliver them to the E2 subunit Regulation of pyruvate dehydrogenase activity Acetyl-CoA has three metabolic fates: 1. It can be metabolized by the citrate cycle, ultimately converting redox energy to ATP by oxidative phosphorylation 2. It can be used to store metabolic energy in the form of fatty acids, which are transported to adipose tissue (fat cells) as triglycerides 3. It is a building block for the synthesis of membrane lipids, cholesterol, and ketones The pyruvate dehydrogenase reaction is essentially irreversible, because physiologic concentrations f reactants and products cannot overcome the biochemical standard free energy change (G = -33.4 kJ/mol), S o production of acetyl-CoA by the pyruvate dehydrogenase reaction is tightly controlled to coordinate the energy needs of the cell with the production of acetyl-CoA The catalytic activity of the pyruvate dehydrogenase complex is primarily regulated by phosphorylation, with the phosphorylated form of pyruvate dehydrogenase being the inactive conformation High acetyl-CoA levels in the mitochondrial matrix, which result from reduced flux through the citrate cycle, reduce the efficiency of the E2 (dihydrolipoyl acetyltransferase) complex by competing with CoA binding 10.3 Enzymatic reactions of the citrate cycle The presence of oxygen is necessary because continual oxidation of NADH and FADH2 inside mitochondria by the electron transport system is required to maintain flux through the citrate cycle The three reactions catalyzed by citrate -31.4 Step 1 Step 5 -2.9 synthase, isocitrate dehydrogenase, and aketoglutarate dehydrogenase are highly favorable, and they are probably even more +6.7 Step 2 Step 6 +0.4 favorable under actual conditions Oxaloacetate concentrations are kept very low in the cell by its conversion to citrate in reaction 1, which allows reaction 8 to proceed -20.9 Step 3 Step 7 -3.8 in the forward direction even though the biochemical standard free energy change is -33.5 Step 4 Step 8 +29.3 large and positive 1. 2. 3. 4. 5. 6. 7. 8. Condensation reaction Isomerization reaction Oxidative decarboxylation Oxidative decarboxylation Phosphorylation Reduction Hydration reaction Redox reaction pyruvate HC Coo pyruvate dehydrogenase CoA + NAD+ acetyl-CoA CO2 + NADH + H+ S CoA 40 Coo oxaloacetate 8 4 malate dehydrogenase HO C H 0 CoA 1 CH citrate synthase Coo COO malate Coo NADH + H+ H NAD+ Ho CHz 7 H2O citrate Coo Hz Coo 2 doo aconitase fumarase H2O 900 fumarate CH HC Coo Yoo isocitrate HO Yoo FADH2 NAD+ CO2 + NADH + H+ FAD 6succinate dehydrogenase coo Coo GTP + CoA GDP + P ssuccinyl-CoA synthetase coo CHz GHz CO S CoA 3 isocitrate dehydrogenase CoA + NAD+ CO2 + NADH + H+ a-ketoglutarate dehydrogenase 4 a-ketoglutarate doo The eight reactions of the citrate cycle Reaction 1: condensation of oxaloacetate and acetyl-CoA by citrate synthase to form citrate The purpose of the first reaction in the citrate cycle is to commit the acetate unit of acetyl-CoA to oxidative decarboxylation Coo do H2O s con e oxaloacetate 900 CoA Ho citrate synthase Écoo CHz acetyl-CoA Coo citrate The citrate synthase reaction follows an ordered mechanism in which oxaloacetate binds first, inducing a conformational change in the enzyme that facilitates acetyl-CoA binding The highly favorable change in biochemical standard free energy of this reaction is due to hydrolysis of the thioester bond in citryl-CoA, which drives the reaction forward The open conformation of the citrate synthase dimer promotes oxaloacetate binding, whereas the closed conformation creates the binding site for acetyl-CoA Reaction 2: isomerization of citrate by aconitase to form isocitrate In the first step, the intermediate cis-aconitate is formed by a dehydration reaction requiring the participation of an iron-sulfur cluster (4 Fe - 4 S) in the enzyme active site In the second step, water is added back (hydration) to convert the double bond in cis-aconitate to a single bond with a hydroxyl group on the terminal carbon COO Hz HO H2O Hik Coo CHz too citrate Coo H2O Éoo coo aconitase aconitase coo cis-aconitate 0 o isocitrate The iron-sulfur cluster in aconitase coordinates the OH group during its removal in the first step, and then facilitates the subsequent hydration reaction There are actually two forms of aconitase: the mitochondrial form that catalyzes reaction 2 in the citrate cycle, and a cystolic form that generates isocitrate for other metabolic pathways Reaction 3: oxidative decarboxylation of isocitrate by isocitrate dehydrogenase to form a-ketoglutarate, CO2, and NADH NAD+ triggers an oxidation reaction that generates the transient intermediate oxalosuccinate, which is then decarboxylated to form the product a-ketoglutarate Coo Coo NAD(P)+ NAD(P)H + H+ iii O HO TH isocitrate dehydrogenase doo isocitrate COO CO2 H+ CH isocitrate dehydrogenase doo doo oxalosuccinate a-ketoglutarate The isocitrate dehydrogenase reaction is favorable under standard conditions (G = -20.9 kJ/mol) and is likely irreversible under normal cellular conditions The isocitrate dehydrogenase reaction is considered to be the ratelimiting step in the cycle and, as such, is highly regulated by ADP and Ca2+ (positive effectors) and ATP and NADH (negative effectors) Reaction 4: oxidative decarboxylation of a-ketoglutarate by a-ketoglutarate dehydrogenase to form succinylcholine-CoA, CO2, and NADH In the first step, a-ketoglutarate binds to the E1 subunit of aketoglutarate dehydrogenase, followed by decarboxylation and formation of a TPP-linked intermediate The a-ketoglutarate dehydrogenase reaction has a large change in biochemical standard free energy and is allosterically regulated in response to changes in the energy charge of the cell Coo Hz I CO2 Coo CoA TPP Lipoamide NAD+ NADH + H+ FAD a-ketoglutarate dehydrogenase complex a-ketoglutarate IS CoA succinyl-CoA Reaction 5: conversion of succinyl-CoA to succinate by succinyl-CoA synthetase in a substrate-level phosphorylation reaction that generates GTP The available free energy in the thioester bond of succinyl-CoA (G = -34 kJ/mol) is used in the succinyl-CoA synthetase reaction to carry out a phosphoric transfer leading to the production of GTP and succinate Coo CHz P CoA GDP GTP Coo Hz succinyl-CoA synthetase s CoA succinyl-CoA succinate In the first step, inorganic phosphate replaces CoA to generate succinylphosphate, which retains the high energy of hydrolysis present in the succinyl-CoA substrate Next, the phosphoryl group is transferred to a nearby His residue to create phosphohistidine, which then passes the phosphoryl group to GDP (or ADP) in the final step The mechanism for the succinyl-CoA synthetase reaction has been compared to the childhood game "pass the hot potato" because the high potential energy available in the thioester bond of succinyl-CoA must be converted to two separate high-energy intermediates before it can be used to phosphorylate GDP Reaction 6: oxidation of succinate by succinate dehydrogenase to form fumarate This pair (FAD/FADH2) is covalently linked to the enzyme succinate dehydrogenase, a protein associated with the inner mitochondrial membrane The succinate dehydrogenase reaction was critical in Hans Kreb s elucidation of the citrate cycle because he found that by inhibiting succinate dehydrogenase with malonate, a reversible inhibitor of the enzyme, he could measure an increase in the accumulation of all five cycle intermediates that precede succinate oxidation 900 FAD doo FADH2 succinate dehydrogenase 00 Reaction 7: hydration of fumarate by fumarase to form malate Fumarase is a highly stereospecific enzyme that catalyzes the hydration of the double bond in fumarate to generate the L isomer of malate OH- 1 Ho fumarase doo Coo H+ fumarase no H doo fumarate carbanion intermediate malate Reaction 8: oxidation of malate by malate dehydrogenase to form oxaloacetate In the process, malate dehydrogenase reduces NAD+ to form the third and final NADH molecule generated by the citrate cycle A histidine residue abstracts a proton from malate to the nicotinamide ring of NAD+ to form NADH COO COO NAD+ NADH + H+ Ho doo malate malate dehydrogenase d o the doo oxaloacetate 10.4 Regulation of the citrate cycle In addition to substrate availability, two other regulatory mechanisms operate in the citrate cycle: product inhibition and feedback control of key enzymes The three main control points within the citrate cycle are regulation of the enzymes citrate synthase (reaction 1), isocitrate dehydrogenase (reaction 3), and aketoglutarate dehydrogenase (reaction 4) Pyruvate dehydrogenase activity is regulated by metabolites that signal the energy charge in the cell and function to control the activity of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase-1 Citrate synthase is potently inhibited by citrate, which is exported out of the mitochondria when its levels are too high 10.5 Metabolism of citrate cycle intermediates The citrate cycle is therefore considered an amphibolic pathway because it functions in both catabolic (oxidation of acetyl-CoA) and anabolic (production of precursors for fatty acid, amino acid, and heme synthesis) pathways Citrate cycle intermediates are shared by other pathways Five citrate cycle intermediates serve as biosynthetic precursors in other pathways: citrate, a-ketoglutarate, succinyl-CoA, malate, and oxaloacetate Acetyl-CoA is used in the cytosol for fatty acid and cholesterol biosynthesis, while oxaloacetate can be converted to malate by cytosolic malate dehydrogenase and shuttled back into the mitochondria to complete the acetyl-CoA circuit The carbon backbones of the amino acids aspartate and glutamate are derived from oxaloacetate and a-ketoglutarate, respectively, through simple transamination reactions Pyruvate carboxylase catalyzes the primary anaplerotic reaction Pyruvate carboxylase contains two active sites: (1) an active site to carboxylate the coenzyme biotin in an ATP-dependent reaction forming carboxybiotin and (2) an active site to carboxylate pyruvate to form oxaloacetate