The Citric Acid Cycle - Succinyl-CoA Synthetase

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Fermentations Pyruvate pyruvate dehydrogenase complex Krebs’ Cycle How did he figure it out? Overview 8 Steps Citrate Synthase Aconitase Isocitrate dehydrogenase Ketoglutarate dehydrogenase complex Succinyl-CoA synthetase Succinate dehydrogenase Fumarase Malate dehydrogenase Energetics Regulation Summary The Citric Acid Cycle Citrate Synthase ✓ ✓ ✓ Aconitase Isocitrate dehydrogenase a-ketoglutarate DHC glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) 1 The Citric Acid Cycle: Succinyl-CoA Synthetase • This step was not appreciated in original cycle until discover of CoA and its role (Fritz Lipmann). Named for the reverse reaction. • Substrate-level phosphorylation (like GAPDH + 1,3-BPG kinase) • The energy of thioester allows for incorporation of inorganic phosphate. • Goes through a phospho-enzyme intermediate • Produces GTP, which can be converted to ATP • Slightly thermodynamically favorable/reversible (DG°’ = –0.7 kcal/mol). – product concentration kept low to pull forward The Citric Acid Cycle: Succinyl-CoA Synthetase Mechanism Phospho-His transfer to NDP; slightly downhill energetically Phosphorolysis: Thioester to mixed anhydride; uphill energetically ❶ Nucleophilic additionelimination ❷ Phosphocreatine Phosphoryl transfer to His; downhill energetically GAPDH 2 The Citric Acid Cycle Citrate Synthase Aconitase ICDH & aKGDH Suc-CoA Synthetase ✓ ✓ ✓ ✓ glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) The Citric Acid Cycle: Succinate Dehydrogenase • Have not seen this chemistry yet: use in the alkane à alkene oxidation. Most often it uses FAD as a cofactor. • But we have seen these next 3 steps. If it worked once, it will work again: Aconitase and ICDH • Famous competitive inhibitor: malonate (OMSGAP) • Reduction requires FADH2 (generally true for alkane to alkene oxidation) – – – – Reduction potential of carbon-hydrogen bond is too low for production of NADH. The 2 hydrogens are removed stereo-specifically. FAD is covalently bound at His, unusual Has a series of 3 iron-sulfur clusters (for re-oxidation of FADH2 by transfer of electrons to electron-transport chain; more later) • Bound to mitochondrial inner membrane – A.K.A. Complex II (more later) • Near equilibrium/reversible (DG°’ = –0.5 kcal/mol); [fumarate] kept low 3 The Citric Acid Cycle: Succinate Dehydrogenase Why use FAD, and not NAD+, for this reaction? NAD+ DE°’ = E°’ (reduction) – E°’ (oxidation) DE°’ = E°’ (FAD) – E°’ (Fumarate) NADH + H+ These are REDUCTION values half-– E°’ (Fumarate) DE°’ =forEeach °’ (NAD) reaction = +0.031 V* – (+0.031 V) = –0.320 V – (+0.031 V) = –0.351 V = 0.0 V DG°’ = –n F DE°’ = –(2)(23.06kcalV-1mol-1)(–0.35 V) = +16 kcalmol-1 *They use +0.05 V in problem #19.3 in book) The Citric Acid Cycle: Succinate Dehydrogenase Mechanism Arg OO– ene-olic intermediate H Arg H Arg OO– Glu H trans Thr Thr Thr H H His Fumarate His FADH2 His H H 4 The Citric Acid Cycle Citrate Synthase ✓ ✓ ✓ ✓ ✓ Succinate dehydrogenase Aconitase ICDH & aKGDH Suc-CoA Synthetase glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) The Citric Acid Cycle: Fumarase sp 2 stereo-specific • Stereospecific – Addition of water is always trans and forms L-malate. – OH− adds to fumarate… then H+ adds to the carbanion. – Cannot distinguish between inner carbons, so either can gain –OH • Slightly thermodynamically favorable/reversible (DG°’ = –0.9 kcal/mol). – product concentration kept low to pull reaction forward 5 The Citric Acid Cycle: Fumarase Mechanism ene-olic intermediate H C HO– –OOC COO– H+ C H The Citric Acid Cycle Citrate Synthase Aconitase ICDH & aKGDH Suc-CoA Synthetase SucDH Fumarase ✓ ✓ ✓ ✓ ✓ ✓ glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) 6 The Citric Acid Cycle: Malate Dehydrogenase Oxidation of Alcohol to a Ketone and Regeneration of Oxaloacetate • Final step of the cycle • Regenerates oxaloacetate for citrate synthase • Highly thermodynamically UNfavorable (DG°’ = +7.1 kcal/mol). – Similar to LDH – Reversible – oxaloacetate concentration kept VERY low by citrate synthase • pulls the reaction forward (–7.7 + 7.1 = –0.6) The Citric Acid Cycle Citrate Synthase Aconitase ICDH & aKGDH Suc-CoA Synthetase SucDH Fumarase Malate DH ✓ ✓ ✓ ✓ ✓ ✓ ✓ glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) 7 The Citric Acid Cycle Origin of C-Atoms in CO2 H2C COOH H2C COOH C COOH HC COOH HO H2C COOH Citrate HO DG°’ = -7.7 +3.2 -7.7 -4.5 C H H2C H2C COOH CH2 O COOH Isocitrate C COOH O C SCoA -7.2 -13.7 -0.7 -14.4 C C H C C H 2C C H 2 C C H Malate Succinyl-CoA a-ketoglutarate -2.0 -6.5 COOH CH2 0 -14.4 -0.9 -15.3 Oxaloacetate +7.1 -8.2 each step cumulative Energetics: why the cycle only turns in one direction • Carbons from acetate are red. • All CO2 generated during the citric acid cycle is produced before succinylCoA is made. • In one turn of the citric acid cycle, neither of the acetyl-CoA carbons is lost. • Both of the CO2 molecules lost were present on the oxaloacetate used to begin the cycle. • Overall energetics makes cycle irreversible: –Citrate synthase, IDH, and KDH – what about kinetics? • Nature of a cycle; acts as a unit and goes as fast as the available carbon The Citric Acid Cycle Citrate Synthase Aconitase ICDH & aKGDH Suc-CoA Synthetase Succinate DH Fumarase Malate DH ✓ ✓ ✓ ✓ ✓ ✓ ✓ Pyruvate dehydrogenase Acetyl CoA Krebs Cycle 8 The Citric Acid Cycle Regulation* aconitase *in mammals The Citric Acid Cycle Regulation* •General regulatory mechanism – activated by substrate availability – inhibited by product accumulation (acetyl-CoA, succinyl-CoA, citrate) – Overall products of the pathway are NADH and ATP. • affects all regulated enzymes in the cycle • inhibitors: NADH and ATP • activators: NAD+, ADP, and AMP •Citrate synthase is also inhibited by succinylCoA. Classic feedback inhibition aconitase – α-Ketoglutarate is an important branch point for amino acid metabolism. – Succinyl-CoA communicates flow at this branch point to the start of the cycle. •Regulation of isocitrate dehydrogenase controls citrate levels; important for fatty acid metabolism. Energy Charge. *in mammals – Aconitase is reversible. – Inhibition of IDH leads to accumulation of isocitrate and reverses aconitase. – Accumulated citrate leaves mitochondria and inhibits phosphofructokinase in glycolysis. 9 The Citric Acid Cycle Yield (TCA): Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O à 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+ Yield (from pyruvate): Pyruvate + 4NAD+ + FAD + GDP + Pi + 2 H2O à 3CO2 + 4NADH + FADH2 + GTP + 3H+ From acetyl-CoA: • Net oxidation of two carbons to CO2 NADH Pyruvate CO2 – equivalent to two carbons of acetylCoA – but NOT the exact same carbons • Energy captured by electron transfer to NADH and FADH2 • Generates 1 GTP, which can be converted to ATP • Cycle acts as a unit; its basically a furnace for burning carbon 3 6 3 3 3 3 ….....except we don’t have the water yet! The Citric Acid Cycle: Summary 4Fe4S TPP Lipoic acid FAD Mn2+ 10 NAD+ FeS isocitrate Mn+2 FAD NAD+ This slide has everything you need to commit to memory NAD+ Pi TPP Lipoic acid FAD 8 steps 8 enzymes 9 structures 13 cofactors /co-substrates Pyruvate Oxidation & Citric Acid Cycle Summary We learned that: • a large multi-subunit enzyme, pyruvate dehydrogenase complex, converts pyruvate into acetyl-CoA • several cofactors are involved in reactions that harness the energy from pyruvate • the citric acid cycle is an important catabolic process: it makes reduced cofactors (NADH & FADH2), plus GTP, that could yield ATP • the rules of organic chemistry help to rationalize reactions in the citric acid cycle • the citric acid cycle is largely regulated by availability of substrates and product inhibition (especially NADH and ATP) 1 Oxidative Phosphorylation Oxidative Phosphorylation Learning goals: • Function of electron-transport chain in mitochondria…... make water (finish the reaction: C6H12O6 + 6O2 à 6CO2 + 6H2O) • Building up the proton-motive force • Synthesis of ATP in mitochondria, chloroplasts, & bacteria • Fuels for the cell, in the form of reduced carbon compounds (sugars), have been burned to carbon dioxide. • Electrons from reduced fuels are transferred to cofactors, which get reduced as NADH or FADH2. • In oxidative phosphorylation, energy from NADH …... But how? and FADH2 is used to make ATP. 12 Oxidative Phosphorylation Glycogenolysis Glycolysis Pyruvate Oxidation Krebs' Cycle Phosphorylation Oxidative 13