Pyruvate dehydrogenase complex Miklós Csala carbohydrates proteins lipids glucose aminoacids fatty acids glycolysis acyl-CoA synthetase e- pyruvate acyl-CoA cytosol resp. chain H+ pyruvate acyl-CoA - H+ e acetyl-CoA H+ - O2 H2O e ADP + Pi citrate - H+ cycle e CO ATP ATP 2 mitochondrium synth. Glycolysis: the net reaction + C6H12O6 + 2NAD + 2ADP + 2Pi - + 2C3H3O3 + 2NADH + 4H +2ATP Mitochondrium outer membrane inner membrane Outer membrane contains pores (composed of porin proteins). These pores are permeable for small molecules so intermembrane space is basically equivalent with the cytosol. Inner membrane is impermeable and small cytosol molecules need transporter proteins to cross. intermembrane cristae space matrix Pyruvate carrier Symport of pyruvate and proton: secondary active transport driven outer by respiration. membrane inner membrane pyruvate pyruvate H+ H+ matrix + cytosol (lower [H ]) (higher [H+]) Oxidative decarboxylation of pyruvate without PDC + H -2H oxidation ionization -H+ CO2 H2O pyruvate acetaldehyde acetaldehyde- acetate hydrate The overall reaction is spontaneous irreversible. Oxidative decarboxylation of pyruvate by PDC Net reaction: pyruvate Some free energy is captured if - CH3-CO-COO the reaction is catalyzed by PDC: + NAD CoA-SH 1. PDC oxidizes with electron carriers (fuels oxidative phosphorylation) and 2. PDC binds acetic acid to CoA NADH CO2 (creates a high energy CH3-CO-S-CoA thioester bond). acetyl-CoA Acetaldehyde and acetate are bound to prosthetic groups within the complex. PD complex •Three metabolic enzymes and two regulatory enzymes in the complex: E1: pyruvate dehydrogenase (TPP, prosthetic group) E2: dihydrolipoyl transacetylase (lipoic acid, prosthetic group) E3: dihydrolipoyl dehydogenase (FAD, prosthetic group) + (two coenzymes involved: NAD and CoA-SH) PDH kinase PDH phosphatase. • Several (12-24) copies of each enzyme with E2 in the center. • The complex is as big as a ribosome. Prosthetic groups: TPP Susceptible to acidic dissociation that makes it a good nucleophilic reagent (carbo-anion). TPP-containing enzymes: pyruvate dehydrogenase (E1 of PDC) α-ketoglutarate dehydrogenase (E1 of αKGDC) branched ketoacid dehydrogenase (E1 of BKADC) transketolase Prosthetic groups: TPP TPP always carries an aldehyde group in aldol linkage TPP-containing enzymes: pyruvate dehydrogenase (E1 of PDC) α-ketoglutarate dehydrogenase (E1 of αKGDC) branched ketoacid dehydrogenase (E1 of BKADC) transketolase Prosthetic groups: lipoic acid Disulfide can be reduced to di- thiol, which can form thioester. Forms amide bond with ε- amino group of a Lysine residue lipoic acid lipoamide (prosthetic group) dihydrolipoic acid Enzymes containing lipoic acid: E2 (transacylase) in PDC, αKGDC and BKADC complexes Prosthetic groups: lipoic acid E1: pyruvate dehydrogenase decarboxylation E2: dihydrolipoyl transacetylase TPP carboanion oxidation transfer E2: dihydrolipoyl transacetylase again transfer E3: dihydrolipoyl dehydrogenase (re-)oxidation transfer of electrons onto a mobile carrier NADH + H+ NAD+ Reaction cycle of PDC Lysyl lipoamide groups act as extended swinging arms between TPPs and FADs. What happens with NADH and acetyl-CoA? NADH All NADH produced in the mitochondrial matrix fuels respiration (and oxidative phosphorylation). There is no other reaction that significantly re-oxidizes NADH in this compartment. Hence PDC reaction is coupled to respiration and can only function in aerobic conditions (although it does not use oxygen directly). acetyl-CoA It can be further oxidized in citrate cycle or used for lipid biosynthesis but it can not be converted to carbohydrates (e.g. glucose) effectively. Unlike other irreversible reactions, PDC step can not be undone. Regulation of PDC Allosteric regulation: Increased levels of acetyl-CoA and NADH inhibit E2, E3. PDC Pyruvate dehydrogenase (E1) + Dihydrolipoyl transacetylase (E2) - + Dihydrolipoyl dehydrogenase - (E3) Pyruvate + HS-CoA + NAD+ NADH + acetyl-CoA + CO2 Regulation of PDC Covalent modification: phosphorylation/dephopsphorylation of E1. NAD+, pyruvate ADP, AMP NADH, acetyl-KoA ATP - + ADP Pyruvate PDC-kinase Pyruvate dehydrogenase dehydrogenase (E1) (E1) P Dihydrolipoyl PDC-phosphatase Dihydrolipoyl transacetylase transacetylase (E2) + (E2) Dihydrolipoyl Pi H2O Dihydrolipoyl dehydrogenase dehydrogenase (E3) Ca2+ (E3) Active Insulin Inactive Regulation of PDC [NADH ] [ATP] [acyl CoA] Activated by low , low , low , [NAD ] [ADP] [CoA] insulin and Ca2+ (exercise in muscle) [NADH ] [ATP] [acyl CoA] Inhibited by high , high , high [NAD ] [ADP] [CoA] PDC facts • Irreversible conversion of pyruvate to acetyl-CoA (oxidative decarboxylation loading NAD+ with electrons). • Converts a carbohydrate precursor to a compound that can be either degraded or used for lipid (fatty acid, cholesterol) and ketone body synthesis. • In mitochondrial matrix (present in every cell that contains mitochondria). • The complex can only function in AEROBIC conditions. • Three metabolic enzymes and two regulatory enzymes in the complex: E1: pyruvate dehydrogenase (TPP, prosthetic group) E2: dihydrolipoyl transacetylase (lipoic acid, prosthetic group) E3: dihydrolipoyl dehydogenase (FAD, prosthetic group) (two coenzymes involved: NAD+ and CoA-SH) PDH kinase PDH phosphatase. PDC-like complexes in mitochondrial matrix E1 and E2 are different (specific to the substrate) but E3 is identical. α-ketoglutarate DH (citrate cycle) CoA CO2 NAD+ NADH α-ketoglutarate succinyl-CoA branched chain α-ketoacid DH (Val, Leu, Ile catabolism) CoA CO2 NAD+ NADH branched α-ketoacid branched acyl-CoA Metabolic diseases related to PDC Beriberi Hypovitaminosis B1 (thiamine) Frequent in Far-East (little thiamine content of rice). in alcoholics (malnutrition). High [pyruvate] and [α-ketoglutarate] in blood. Neurologic and cardiac symptomes. Can be diagnosed by RBC transketolase assay. Genetic defects of PDC E1 or E2 defect: lactic acidosis (and high [pyruvate]) severe neurologic defects in newborn (due to affected ATP and lipid synthesis). E3 defect: the same plus citrate cycle defect and demaged branched chain amino/keto-acid catabolism Citrate cycle Miklós Csala carbohydrates proteins lipids glucose aminoacids fatty acids glycolysis acyl-CoA synthetase e- pyruvate acyl-CoA cytosol resp. chain H+ pyruvate acyl-CoA - H+ e acetyl-CoA H+ - O2 H2O e ADP + Pi citrate - H+ cycle e CO ATP ATP 2 mitochondrium synth. Alternative names and history Citrate cycle (or citric acid cycle) is also referred to as tricarboxylate cycle (TCA cycle), Szent-Györgyi cycle (by proud Hungarians) Krebs cycle (by non-Hungarians) Szent-Györgyi-Krebs cycle (by modest Hungarians). In 1935, Szent-Györgyi demonstrated that little amounts (catalytic amounts) of some dicarboxylates (succinate, fumarate, malate or oxaloacetate) acelerate respiration, and showed the following interconversions: succinate → fumarate → malate → oxaloacetate. In 1936, Martius and Knoop reported these reactions: citrate → cis-aconitate → isocitrate → ketoglutarate → succinate. In 1937, Krebs found the missing reaction and the cycle was closed. „Enzymatic conversion of pyruvate + oxaloacetate to citrate and CO2” means PDC reaction and the entry of acetyl-CoA into the cycle. He showed that the cycle is a major pathway for pyruvate oxidation in muscle. Overall citrate cycle Citrate synthase IRREVERSIBLE Overall ΔGo’ = -31.5 kJ/mol. Aconitase Isocitrate dehydrogenase Oxidative decarboxylation IRREVERSIBLE α-Ketoglutarate dehydrogenase complex Oxidative decarboxylation IRREVERSIBLE The complex is very similar to PDC and contains the same E3. Succinyl-CoA synthetase Substrate level phosphorylation GTP can pass the phosphoryl group to ADP (NDP kinase). Succinate dehydrogenase Membrane bound flavoprotein. Part of succinate : CoQ reductase complex (Complex II of respiratory chain). The two electrons are transferred to CoQ by Fe-S proteins of the complex. Succinate dehydrogenase Malonate competitively inhibits succinate dehydrogenase. succinate malonate oxalate Fumarase (Stereospecific hydration: always L-malate is formed.) Malate dehydrogenase This reaction is not spontaneous in standard conditions: ΔGo’ = +29.7 kJ/mol. It is pushed forward by high [malate]/[oxaloacetate] and [NAD+]/[NADH] ratios. Overall citrate cycle Because of the stereochemistry, the two carbon atoms lost as CO2 during phase I are not the same as those introduced as acetyl-CoA. Strictly: one “acetyl equivalent” is oxidized to two molecules of CO2 in each round. Net reaction of citrate cycle + CH3CO-S-CoA + 3NAD + FAD + GDP + Pi + 2H2O + 2CO2 + HS-CoA + 3NADH + 2H + FADH2 + GTP Citrate cycle facts • Irreversible oxidation of acetyl group (of acetyl-CoA) to 2CO2 + (oxidation loading NAD and FAD with electrons). • In mitochondrial matrix (present in every cell that contains mitochondria). • NADH and FADH2 generated in the cycle can only be reoxidized by the respiratory chain. • Hence the cycle can only function in AEROBIC conditions, although it does not use oxygen directly. • The intermediates of the cycle are neither produced nor consumed during the reaction (they are always regenerated). They can rather be considered as catalytic cofactors. • The intermediates can be consumed by other (anabolic) processes, so they must be resynthesized (anaplerosis). Standard and physiological ΔG ΔGo’ ΔG Rxn Enzyme (kJ/mol) (kJ/mol) 1 Citrate synthase -31.5 negative 2 Aconitase ≈ 5 ≈ 0 3 Isocitrate dehydrogenase -21 negative 4 α-Ketoglutarate dehydrogenase complex - 33 negative 5 Succinyl-CoA synthetase -2.1 ≈ 0