Citric Acid Cycle PDF

Topic 6 THE CITRIC ACID CYCLE November 16, 2021 The Citric Acid Cycle Oxidative Decarboxylation The Citric Acid Cycle Van Nguyen 1  Metabolism Carbohydrates Metabolism Energy and electron transfer Lipids Metabolism...

Topic 6 THE CITRIC ACID CYCLE November 16, 2021 The Citric Acid Cycle Oxidative Decarboxylation The Citric Acid Cycle Van Nguyen 1  Metabolism Carbohydrates Metabolism Energy and electron transfer Lipids Metabolism Summary of Energetic pathways in humans Viel & Gaudet, Principle of Biochemistry, 2015 Van Nguyen 2  Acetyl CoA is the Main Entry Point to the Citric Acid Cycle Under aerobic conditions, pyruvate enters the mitochondria where it is converted into acetyl CoA. Acetyl CoA is the fuel for the citric acid cycle, which processes the two-carbon acetyl unit to two molecules of CO2 while harvesting high-energy electrons that can be used to form ATP. Mitochondrion has a double membrane TCA cycle takes place within the matrix. Van Nguyen 3 Citric acid cycle Oxidative decarboxylation Citric acid cycle Van Nguyen 4  Part A: Oxidative decarboxylation 3 Steps Pyruvate Dehydrogenase complex PDH 3 enzymes 5 coenzymes Irreversible 2 regulatory enzymes Van Nguyen 5 Pyruvate dehydrogenase complex and coenzymes 3 enzymes in complex (noncovalently assemble): 5 E1: pyruvate For dehydrogenase (cat.) For E2: dihydrolipoyl (sub.) transacetylase (cat.) For E3: dihydrolipoyl (cat.) dehydrogenase (sub.) a component of the cat.: catalyst: in-out mitochondrial matrix sub.: substrate: spent PDH regulatory enzymes PDK: pyruvate dehydrogenase kinase PDP: pyruvate dehydrogenase phosphatase Van Nguyen 6 Reactions: 3 steps Van Nguyen 7  Steps of oxidative decarboxylation 1) Decarboxylation pyruvate dehydrogenase (E1) R’ R Van Nguyen 8 Mechanism of the E1 Decarboxylation Reaction 1.TPP forms a carbanion. 2.The carbanion attacks the carbonyl group of pyruvate. 3.Decarboxylation occurs. The positive charge on the TPP stabilizes the negative charge resulting from the decarboxylation. 4.Protonation occurs to yield the hydroxyethyl-TPP intermediate. Van Nguyen 9 Steps of oxidative decarboxylation pyruvate dehydrogenase E2 E2 R’’ Van Nguyen 10 Steps of oxidative decarboxylation dihydrolipoyl transacetylase (thioester: high energy) Van Nguyen 11 Thioester: high energy Van Nguyen 12  Steps of oxidative decarboxylation of lipoamide dihydrolipoyl dehydrogenase Regenerate of FAD Generate NADH (oxidized) Van Nguyen 13 Summary of decarboxylation steps TPP in Lipoamide in in Catalyzed by E1 Catalyzed by E1 Catalyzed by E2 TPP out Reduced Lipoamide out NADH generation NAD+ in FAD in Catalyzed by E3 FAD regeneration FADH2 out Lipoamide out Van Nguyen 14 Mechanism at PDH sites NADH NAD+ Acetyl CoA CoA Van Nguyen 15 Flexible Linkages Allow Lipoamide to Move Between Different Active Sites The three enzymes of the pyruvate dehydrogenase complex are structurally integrated, and the lipoamide arm allows rapid movement of substrates and products from one active site of the complex to another. Dihydrolipoamide is formed by the attachment of the vitamin lipoic acid to a lysine residue in dihydrolipoyl transacetylase (E2). The core of the pyruvate dehydrogenase complex is formed by 60 molecules of E2, the transacetylase. Van Nguyen 16 Structure of the Pyruvate Dehydrogenase Complex from B. stearothermophilus The image of the complex, which was derived from cryo-electron microscopic data (Section 3.5), shows an inner core consisting of the E2 enzyme. The shell surrounding the core consists of E1 and E3 enzymes, although only the E1 enzymes are shown in this structure. Two of the 60 lipoamide arms are shown (red and yellow) Van Nguyen 17 Structure of the Transacetylase (E2) Core The figure shows one subunit of the transacetylase trimer. Notice that each subunit consists of three domains: a lipoamide-binding domain, a small domain that interacts with E3, and a large transacetylase catalytic domain. The catalytic domains interact with one another to form the catalytic trimer. Transacetylase domains of three identical subunits are shown, with one depicted in red and the others in white in the ribbon representation. Van Nguyen 18 Regulation of PDH PDH regulatory enzymes PDK: pyruvate dehydrogenase kinase PDP: pyruvate dehydrogenase phosphatase Van Nguyen 19 Coenzymes TPP Thiamine pyrophosphate (TPP) assists in the decarboxylation of α-keto acids (and in the formation and cleavage of α-hydroxy ketones as in the transketolase reaction). Van Nguyen 20 Coenzymes NAD+ The Nicotinamide Coenzymes – NAD+/NADH and NADP+/NADPH - carry out hydride (H:-) transfer reactions. All reactions involving these coenzymes are two-electron transfers. Van Nguyen 21 Coenzymes FAD The Flavin Coenzymes – FAD/FADH2 and FMN/FMNH2 Van Nguyen 22 Coenzymes FAD Flavin coenzymes can exist in any of three oxidation states, and this allows flavin coenzymes to participate in one-electron or two-electron transfer reactions. Partly because of this, flavoproteins catalyze many reactions in biological systems and work with many electron donors and acceptors. Van Nguyen 23 Coenzymes A The two main functions of Co A are: 1) Activation of acyl groups for transfer by nucleophilic attack 2) Activation of the α-hydrogen of the acyl group for abstraction as a proton The reactive sulfhydryl group on CoA mediates both of these functions. The sulfhydryl group forms thioester linkages with acyl groups. Van Nguyen 24 Coenzymes Lipoic acid Lipoic acid functions to couple acyl-group transfer and electron transfer during oxidation and decarboxylation of α-keto acids. It is found in pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Lipoic acid is covalently bound to relevant enzymes through amide bond formation with the ε-NH2 group of a lysine side chain. Van Nguyen 25  Part B: Citric acid cycle Steps Control/regulation Different fates of Acetyl-CoA’s carbon atoms Biosynthetic roles of TCA cycle “Filling up” reactions Diseases Van Nguyen 26 Citric acid cycle 2C Citric acid cycle Tricarboxylic acid cycle (TCA) 4C 6C Krebs cycle 6C The citric acid cycle is amphibolic; 4C that is, it plays a role in both 5C catabolism and anabolism 4C 4C 4C Van Nguyen 27 Van Nguyen 28 The Enzymes and Reactions of the TCA Cycle Van Nguyen 29 The citrate synthase reaction 1 Cα of the acetyl group in acetyl-CoA is acidic and can be deprotonated to form a carbanion This carbanion is a strong nucleophile that can attack the α-carbonyl of oxaloacetate, yielding citryl-CoA Thioester hydrolysis then produces citrate DG°’ = -32.8 kJ mol-1, therefore, the reaction is exergonic Citrate synthase enzyme is allosterically inhibited by NADH, ATP, and succinyl-CoA Van Nguyen 30 Mechanism: The Mechanism of Citrate Synthase Prevents Undesirable Reactions Citrate synthase exhibits induced fit, since oxaloacetate binding induces structural changes in the enzyme that lead to the formation of the acetyl CoA binding site. – This also indicates that it is an example of “ordered sequential” kinetics. The formation of the reaction intermediate citryl CoA causes a dramatic structural change that completes active site formation, enabling cleavage of the thioester linkage. Citryl CoA is then cleaved to form citrate and CoA. Van Nguyen 31 Conformational Changes in Citrate Synthase on Binding Oxaloacetate Van Nguyen 32 Aconitase reaction 2 Citrate: tertiary alcohol, poor substrate for oxidation Isocitrate: secondary –OH, readily oxidized Aconitase is stereospecific and removes the pro-R hydrogen from the pro-R arm of citrate. Van Nguyen 33  Binding of Citrate to the Iron–Sulfur Complex of Aconitase Aconitase, an iron–sulfur protein (also referred to as a non-heme iron protein), catalyzes the formation of isocitrate from citrate. A 4Fe-4S iron–sulfur cluster is a component of the active site of aconitase. Notice that one of the iron atoms of the cluster binds to a COO− group and an OH group of citrate. Van Nguyen 34 Isocitrate Dehydrogenase reaction 3 With isocitrate dehydrogenase, C-2 alcohol of isocitrate is oxidized by NAD+ !-decarboxylation reaction à CO2 Van Nguyen 35 !-Ketoglutarate Dehydrogenase reaction 4 Nearly identical to PDH structurally and mechanistically α-ketoglutarate dehydrogenase complex consists of α-ketoglutarate dehydrogenase dihydrolipoyl transsuccinylase dihydrolipoyl dehydrogenase Five coenzymes used - TPP, CoASH, lipoic acid, NAD+, and FAD Van Nguyen 36 Succinyl-CoA Synthetase reaction 5 Hydrolysis of succinyl-CoA (a CoA ester) drives the phosphorylation of GDP to produce GTP Van Nguyen 37 Mechanism of Succinyl- CoA Synthetase reaction Phosphate anion carries out a nucleophilic attack on the carbonyl C of succinyl-CoA, forming succinyl phosphate Subsequent phosphoryl transfer to an active-site His forms a phosphohistidine intermediate, releasing the product succinate The phosphoryl group is then transferred to GDP to produce GTP Van Nguyen 38 ATP or GTP May be Produced at this Step In mammals, there are two isozymic forms of the enzyme, one specific for ADP and one for GDP. – In tissues that perform large amounts of cellular respiration (e.g., skeletal and heart muscle), the ADP-requiring isozyme predominates. – In tissues that perform many anabolic reactions (e.g., liver), the GDP-requiring enzyme is common and can work in reverse so that GTP is used to power succinyl CoA synthesis. Succinyl CoA is then used in heme synthesis. If one NTP accumulates but the other is needed, they can be readily interconverted by nucleoside diphosphokinase. Van Nguyen 39 Oxidation of Succinate to Oxaloacetate This process involves a series of three reactions These reactions include: Oxidation of a C-C single bond to a double bond Hydration across the double bond Oxidation of the resulting alcohol to a ketone This trio of reactions will be seen again in: Fatty acid breakdown and synthesis Amino acid breakdown and synthesis Van Nguyen 40  The Succinate Dehydrogenase Reaction 6 The mechanism involves hydride removal by FAD and a deprotonation This enzyme is actually part of the electron transport pathway in the inner mitochondrial membrane The electrons transferred from succinate to FAD (to form FADH2) are passed directly to ubiquinone (UQ) in the electron transport pathway Van Nguyen 41 The Fumarase Reaction 7 Fumarase Catalyzes the trans-Hydration of Fumarate to Form L-Malate. The actual mechanism is not known for certain Van Nguyen 42 The Malate Dehydrogenase Reaction 8 This reaction is energetically expensive: "Go' = +30 kJ/mol Thus the concentration of oxaloacetate in the mitochondrial matrix is quite low However, the malate dehydrogenase reaction is pulled forward by the favorable citrate synthase reaction Van Nguyen 43 Control of the Citric Acid Cycle There are 3 points of control within the cycle: 1 Citrate synthase: inhibited by ATP, NADH, and succinyl CoA; also product inhibition by citrate 3 Isocitrate dehydrogenase: activated by ADP and NAD+, inhibited by ATP and NADH 4 a-ketoglutarate dehydrogenase complex: inhibited by ATP, NADH, and succinyl CoA; activated by ADP and NAD+ There is one control point outside the cycle Pyruvate dehydrogenase: inhibited by ATP and NADH; also product inhibition by acetyl-CoA Van Nguyen 44 Control of the Citric Acid Cycle Van Nguyen 45 Energetics of the Citric Acid Cycle The TCA cycle is exergonic, net !Gº' of -40 kJ/mol for one pass around the cycle Van Nguyen 46  Summary Oxidation of Pyruvate Forms CO2 and ATP Van Nguyen 47  Summary Oxidation of Pyruvate Forms CO2 and ATP Each pair of electrons from NADH will generate ~2.5 ATP when used to reduce oxygen in the electron-transport chain. Each pair of electrons from FADH2 will power the synthesis of ~1.5 ATP with the reduction of oxygen in the electron-transport chain. 1 glucose à 2 NADH + 2 ATP + 2 pyruvate = 5 ATP + 2 ATP + 25 ATP = 32 ATP Van Nguyen 48 The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle The carbonyl C of acetyl-CoA becomes CO2 only in the second turn of the cycle (following entry of acetyl-CoA ) The methyl C of acetyl-CoA survives two cycles completely, but half of what's left exits the cycle on each turn after that The C-C bond cleaved in a given TCA cycle actually entered as an acetate in the previous turn Thus the oxidative decarboxylations that cleave this bond are just a disguised acetate C-C cleavage and oxidation Van Nguyen 49  The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle The carboxyl carbon atom of acetate is released as CO2 in the second turn of the cycle Van Nguyen 50 The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle Release of the methyl-C of acetate as CO2 requires multiple turns of the cycle. 51 Can the TCA Cycle Provide Intermediates for Biosynthesis? The TCA cycle provides several of these #-Ketoglutarate can be transaminated to make glutamate, which can be used to make purine nucleotides, as well as Arg and Pro Succinyl-CoA can be used to make porphyrins Fumarate and oxaloacetate can be used to make several amino acids and also pyrimidine nucleotides Note that mitochondrial citrate can be exported to be a cytoplasmic source of acetyl-CoA and oxaloacetate Van Nguyen 52 Biosynthetic Roles of the Citric Acid Cycle 53 The TCA Cycle Can Provide Intermediates For Biosynthesis The TCA cycle provides intermediates for biosynthesis. Amino acids are highlighted in orange. All 20 common amino acids can be made from metabolites derived from the TCA cycle. Van Nguyen 54 The Citric Acid Cycle Must be Capable of Being Rapidly Replenished Citric acid cycle intermediates must be replenished if any are used for anabolic reactions. Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate. Instead, oxaloacetate is formed by the carboxylation of pyruvate; this reaction is catalyzed by pyruvate carboxylase. Recall that this reaction is also used in gluconeogenesis and is dependent on the presence of acetyl CoA. This is an example of an anapleurotic (“filling up”) reaction, since it replenishes the supply of a critical citric acid cycle intermediate. Van Nguyen 55 What Are the Anaplerotic, or “Filling Up,” Reactions? Pyruvate carboxylase - converts pyruvate to oxaloacetate. This is the most important anaplerotic reaction PEP carboxylase - converts PEP to oxaloacetate Malic enzyme converts pyruvate into malate Van Nguyen 56 What Are the Anaplerotic, or “Filling Up,” Reactions? Pyruvate carboxylase (shown here), and also phosphoenolpyruvate (PEP) carboxylase, and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle intermediates. Van Nguyen 57 What Are the Anaplerotic, or “Filling Up,” Reactions? Pyruvate carboxylase , phosphoenolpyruvate (PEP) carboxylase (shown here), and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle intermediates. Van Nguyen 58 What Are the Anaplerotic, or “Filling Up,” Reactions? Pyruvate carboxylase, and also phosphoenolpyruvate (PEP) carboxylase and malic enzyme (shown here) catalyze anaplerotic reactions, replenishing TCA cycle intermediates. Van Nguyen 59 TCA Summary 60 Pyruvate Dehydrogenase Phosphatase Deficiency Individuals with pyruvate dehydrogenase phosphatase deficiency have a pyruvate dehydrogenase complex that is always phosphorylated (i.e., inactive). In these individuals, glucose is processed to lactate rather than to acetyl CoA, and high blood lactic acid results. Many systems malfunction in the acidified environment, particularly the central nervous system. Van Nguyen 61 Defects in the Citric Acid Cycle Contribute to the Development of Cancer The defects in the enzymes succinate dehydrogenase, fumarase, pyruvate dehydrogenase kinase, and isocitrate dehydrogenase contribute to cancer growth. Mutations in the first three of these enzymes activate HIF-1, leading to enhanced aerobic glycolysis. Mutations in isocitrate dehydrogenase result in the synthesis of 2-hydroxyglutarate, which modifies methylation patterns in DNA. These modifications can alter gene expression and promote rapid cell growth. Van Nguyen 62 An Enzyme in Lipid Metabolism is Hijacked to Inhibit Pyruvate Dehydrogenase Activity New findings suggest another metabolic feature of cancer cells. Normally, the mitochondrial enzyme acetyl CoA acetyltransferase synthesizes ketone bodies (e.g., acetoacetate), which serve as a fuel source for various tissues. However, in some cancers, the enzyme becomes phosphorylated, which changes its activity so that it acts as a protein acetyltransferase instead. It can then acetylate both pyruvate dehydrogenase and pyruvate dehydrogenase phosphatase. That acetylation inhibits those enzymes and facilitates the metabolic switch from oxidative phosphorylation to aerobic glycolysis (known as the Warburg effect). Van Nguyen 63 The Disruption of Pyruvate Metabolism is the Cause of Beriberi and Poisoning by Mercury and Arsenic (1/2) Thiamine deficiency results in insufficient activity of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, as well as an enzyme in the pentose phosphate pathway (transketolase). Thymine is the precursor of the cofactor thiamine pyrophosphate (TPP), and its absence prevents the functioning of those enzymes. The neurologic and cardiovascular disorder beriberi can result. The vitamin thiamine is found in brown rice but not in white (polished) rice. Van Nguyen 64 The Disruption of Pyruvate Metabolism is the Cause of Beriberi and Poisoning by Mercury and Arsenic (2/2) Pyruvate dehydrogenase complex activity can also be inhibited by mercury and arsenite, which bind to the two sulfurs of dihydrolipoamide. 2, 3-Dimercaptopropanol can counter the effects of arsenite poisoning by forming a complex with the arsenite that can be excreted. Early hatters (hatmakers) used mercury to make felt, which inhibited activity of the pyruvate dehydrogenase complex in the brain, often leading to strange behavior. Van Nguyen 65 Arsenite Poisoning Van Nguyen 66 Diabetic Neuropathy may Result from Inhibition of the Pyruvate Dehydrogenase Complex Diabetic neuropathy (DN), a numbness, tingling, or pain in the limbs and digits, is a common complication of both type 1 and type 2 diabetes. The symptoms may be caused by overproduction of lactic acid by cells in the dorsal root ganglion, a part of the nervous system responsible for pain perception. Lactate is a common fuel for neurons, which import the lactate and convert it to pyruvate for use in cellular respiration. Van Nguyen 67 Diabetic Neuropathy may Result from Inhibition of the Pyruvate Dehydrogenase Complex The increase in lactic acid in diabetics may be due to hyperglycemia (high glucose), the defining feature of diabetes. This increases pyruvate dehydrogenase kinase activity in the cells of the dorsal root ganglion. This kinase then phosphorylates and inhibits the pyruvate dehydrogenase complex. Glycolytically produced pyruvate is then converted to lactate, and excess lactate leads to an increase in acid-sensing pain receptors. These findings may lead to new therapeutic interventions. Van Nguyen 68 New Treatments for Tuberculosis may be on the Horizon (1/3) The bacterium responsible for tuberculosis (TB), Mycobacterium tuberculosis, is transmitted by people with active lung infections by coughing and sneezing. A common treatment for TB is the antibiotic rifampicin, which acts as an inhibitor of bacterial protein translation. However, as resistant bacterial strains emerge, new treatments are needed. The bacteria are dependent on the glyoxylate cycle (which allows conversion of fats to glucose), especially when they are in a latent state in the lungs. Van Nguyen 69  New Treatments for Tuberculosis may be on the Horizon (2/3) A key enzyme in the glyoxylate cycle is isocitrate lyase. In the process of catalysis, a reactive thiolate is formed on cysteine 191 in the active site. Van Nguyen 70  New Treatments for Tuberculosis may be on the Horizon (3/3) A suicide (or mechanism-based) inhibitor for the lyase has been synthesized. When this inhibitor, 2-vinyl-isocitrate, reacts with the lyase, succinate is released as in the normal reaction. However, the cysteine of the lyase remains covalently modified. Because this cysteine is conserved in all M. tuberculosis strains, the likelihood of evolving resistance is diminished. Van Nguyen 71 References Viel & Gaudet, Principle of Biochemistry, 2015 Garrett & Grisham, Biochemistry, 2017 Campbell & Farrell, Biochemistry, 2016 Lehninger, Principle of Biochemistry, 2013 Berg, Biochemistry, 2019 Van Nguyen 72