3 9 15 Biochemistry 401 Lecture 20 The TCA cycle Eight Reactions Purpose Acquisition of Energy from Fuels Production of Building blocks Anabolic precursors Oxidation Oxidative Decarboxylations Formation of NADH 3 FADH2 1 GTP 1 Key Reactions Key Regulated Steps 3 major points of regulation These Notes are Color Coded Enzymes Inhibitors Positive effectors Important Anabolic Products Key thoughts regarding regulation There are primarily 3 modes of regulation 1 Availability of substrate 2 Product inhibition 3 Competitive feedback by downstream intermediates TCA Cycle Tricarbocxylic Acid Cycle Citric Acid Cycle Krebs Cycle 1 3 9 15 2 3 9 15 3 3 9 15 4 3 9 15 Can you match the type of reaction some letters are used more than once A Redox B Oxidative decarboxylation C Isomerization D Hydration E Substrate level phosphorylation F Condensation 1 Oxaloacetate Acetyl CoA H2O Citrate CoA 2 Citrate Isocitrate 3 4 Isocitrate NAD Ketoglutarate CO2 NADH H Ketoglutarate NAD CoA Succinyl CoA CO2 NADH H 5 Succinyl CoA GDP or ADP Pi Succinate CoA GTP or ATP 6 Succinate FAD Fumarate FADH2 7 Fumarate H2O Malate 8 Malate NAD Oxaloacetate NADH H 5 3 9 15 Can you name the products 1 Oxaloacetate Acetyl CoA H2O 2 3 Citrate Isocitrate NAD 4 Ketoglutarate NAD CoA 5 Succinyl CoA GDP or ADP Pi 6 Succinate FAD 7 Fumarate H2O 8 Malate NAD Reaction 1 Enzyme Citrate Synthase Condensation Reaction forming 6C molecule Formation of Citrate from Oxaloacetate and Acetyl CoA 4C 2C intermediate 6C Condensation driven by CoA hydrolysis 6 3 9 15 Reaction 1 Enzyme Citrate Synthase Condensation Reaction forming 6C molecule Formation of Citrate from Oxaloacetate and Acetyl CoA 4C 2C intermediate 6C Regulation Negative ATP NADH Succinyl CoA Dimer Each monomer has a small and large domain Sequential ordered reaction OAA binds first and causes the small subunit of Citrate Synthase to rotate this forms the Acetyl CoA binding pocket This brings closer the residues that are necessary for the condensation reaction between OAA and Acetyl Example of induced fit CoA to occur High Citrate Levels Indicate a Well Fed State Mitochondria Cytoplasm 7 3 9 15 Reaction 2 Enzyme Aconitase Isomerization in 2 steps A Dehydration of Citrate to yield a C C cis Aconitate B Stereospecific Rehydration of the double bond yields Isocitrate Pro chiral Plane of symmetry Chiral Enzyme Aconitase Reaction 2 Isomerization in 2 parts Why Setting up for the first Redox Reaction Isomerization in 2 steps Dehydration followed by re hydration Puts the hydroxyl group in better position for oxidation and CO2 release in the next step Oxidative Decarboxylation Reaction 3 Enzyme Isocitrate Dehydrogenase A Oxidation reduction reaction NADH H produced B Decarboxylation reaction CO2 ketoglutarate produced 6C 1C 5C Unstable Ketoacid intermediate First reaction in which CO2 is produced NADH is also produced and will be used to make ATP 8 3 9 15 Regulation 1 4 major points of regulation a PDC NAD CoA Acetyl CoA NADH ATP b Citrate Synthase Succinyl CoA NADH ATP c Isocitrate DH NAD ADP NADH ATP d AKG DH AMP Succinyl CoA NADH 2 Individual reactions can be regulated at the level of products and reactants Le Chatelier s Principle Reaction 3 Enzyme Isocitrate Dehydrogenase Reaction 3 Isocitrate Dehydrogenase The first of 4 oxidation reduction reactions CO2 release First of 2 NADH produced ADP decreases KM of the enzyme Isocitrate Rate limiting step Dehydrogenase for its Regulation substrates Positive ADP NAD Negative NADH direct competition with NAD ATP 9 3 9 15 Reaction 4 4th enzyme is an enzyme complex Ketoglutarate Dehydrogenase Complex Addition of CoA and release of CO2 The enzymes are similar to those in Pyruvate Dehydrogenase Complex E1 and E2 are homologous to those in PDH and E3 is identical to PDH E3 Together these enzymes make up the ketoglutarate Dehydrogenase complex The second NADH and CO2 are produced From now on 4C Ketoglutarate Dehydrogenase Complex This enzyme is a complex like the Pyruvate Dehydrogenase Complex E1 and E2 are similar E3 is identical to PDC Reaction 4 Production of Products 2nd NADH 2nd and final CO2 Succinyl CoA used for Heme synthesis Addition of CoA Sets up the production of GTP in the next reaction Regulation Positive AMP Negative Succinyl CoA NADH Reaction 5 Succinyl CoA Synthetase Named for the reverse reaction Substrate level Phosphorylation of GDP Production of GTP Release of CoA 10 3 9 15 Enzyme 5 Succinyl CoA Synthetase named for reverse reaction Release of CoA allows for a energetically favorable transfer of inorganic phosphate to GDP GTP can be used for energy directly can be used to make ATP or can be used in signaling pathways Reactions 6 8 Regeneration of OAA 6 7 OX Oxidation Reduction Reaction FADH2 produced HYD 8 6 Hydration across the double bond 7 sets up the next reaction OX Last Oxidation Reduction Reaction regenerates OAA 8 NADH produced Reaction 6 Enzyme Succinate Dehydrogenase Oxidation number 3 using covalently attached FAD as a cofactor Electrons transferred to iron sulfur clusters in the enzyme and ultimately Oxygen by way of other members of the electron transport chain Production of FADH2 Regulation Negative FADH2 FAD 11 3 9 15 Reaction 6 Enzyme Succinate Dehydrogenase Why FAD Not NAD Not enough energy to reduce NAD a 2 electron hydride addition in Succinate Dehydrogenase Flavin ring is covalently attached Is imbedded in the inner mitochondrial membrane Is also part of the electron transport chain Reaction 7 Enzyme Fumarase Setting up for the next oxidation Addition of H2O across the double bond sets up for the 4th and final oxidation reduction reaction Product Malate Used in Gluconeogenesis 12 3 9 15 H 2O Reaction 7 Enzyme Fumarase H2O added in trans across the double bond of Fumarate to yield LMalate This stereo specific reaction is made possible by Fe S complex in the enzyme Reaction 8 Enzyme Malate Dehydrogenase Malate NAD Oxaloacetate NADH H MNO Reaction 8 Malate Dehydrogenase No matter where you go There you are Buckaroo Banzai Regeneration of Oxaloacetate to re start the cycle Oxidation reaction that produces NADH Regulation Negative NADH NAD G 7Kcal mole rxn must be reversible Drive reaction by removing product OAA 13 3 9 15 Regulation 1 4 major points of regulation a PDC NAD CoA Acetyl CoA NADH ATP b Citrate Synthase Succinyl CoA NADH ATP c Isocitrate DH NAD ADP NADH ATP d AKG DH AMP Succinyl CoA NADH 2 Individual reactions can be regulated at the level of products and reactants Le Chatelier s Principle What
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