TCA Cycle PDF

Citric Acid Cycle Learning Objectives - Understand the significance of Citric Acid Cycle - Explain the eight reaction steps in TCA cycle (enzymes, metabolites, chemical changes, generation of ATP/GTP and NADH/FADH2) - Trace the carbon flow through the cycle...

Citric Acid Cycle Learning Objectives - Understand the significance of Citric Acid Cycle - Explain the eight reaction steps in TCA cycle (enzymes, metabolites, chemical changes, generation of ATP/GTP and NADH/FADH2) - Trace the carbon flow through the cycle - Briefly describe the key regulatory steps and major regulatory molecules - Recognize the cataplerotic reactions and anaplerotic reactions that affect the levels of intermediates in the cycle.  Citric Acid Cycle Citric acid - a natural preservative and a reservoir of carbon in citrus fruits - its accumulation in citrus fruits is not related to the role of citric acid in the citric acid cycle 2 Cellular respiration - Aerobic conditions - Complete oxidation of glucose, amino acids and fatty acids to CO2 and H2O - Three major stages: (1) Acetyl-CoA production (2) Acetyl-CoA oxidation - Citric acid cycle - or Tricarboxylic acid (TCA) cycle - or Kreb cycle (3) Electron transfer and oxidative phosphorylation 3 Entry of pyruvate to mitochondria gluconeogenesis Inner membrane 4 Production of acetyl-CoA from pyruvate - Inside mitochondria (matrix) - A link between glycolysis and TCA cycle - Involves an enzyme complex with 3 components (E1, E2, and E3) - Overall an “oxidative decarboxylation” reaction - Five cofactors are involved (NAD+ as the final oxidizing agent) (Co-enzyme A) 5 6 Pyruvate Dehydrogenase (PDH) Complex - Consisting of 3 different enzymes (E1, E2, E3) catalyzing five reactions - Complex formation avoids diffusion of intermediates and allows efficient metabolite channeling 7 E1: Pyruvate dehydrogenase - Catalyzes two reactions 1. Decarboxylation of pyruvate -TDP is used as a prosthetic group in the active site. Thiamine diphosphate (TDP) 8 E1: Pyruvate dehydrogenase 2. Transfer of acetyl group to lipoamide (a prosthetic group in E2) (a lysine residue in E2) 9 E2: Dihydrolipoamide acetyltransferase 3. Transfer of acetyl group to co-enzyme A: (Fully reduced form) Arsenite poisoning: Inhibition of E2 10 E3: Dihydrolipoamide dehydrogenase - Contains FAD as a prosthetic group 4. Regeneration of lipoamide (oxidized form) 5. Regeneration of FAD (oxidized form) 11 12 Regulation of the pyruvate dehydrogenase (PDH) complex (1) Product inhibition and substrate activation of E2 and E3 13 Regulation of the pyruvate dehydrogenase complex (2) Phosphorylation of E1 (inactivation) 14 Citric acid (or TCA) cycle Mitochondria 15 16 Reactions and enzymes of the citric acid (TCA) cycle (1) Citrate synthase - Condensation of OAA and acetyl CoA (OAA) Citric acid is a Enzyme-bound intermediate tricarboxylic acid (TCA) 17 (2) Aconitase - an isomerase (mutase) 18 (3) Isocitrate dehydrogenase - Catalyzes an oxidative decarboxylation 19 (4) α-Ketoglutarate dehydrogenase complex - An enzyme complex with 3 components (E1, E2, and E3) - Catalyzes an oxidative decarboxylation TPP, Lipoate, FAD - Resembles pyruvate dehydrogenase complex in both structure and function: 20 (5) Succinyl-CoA synthetase - Hydrolysis of the thioester succinyl-CoA - Named for the reverse reaction (ligation of succinate and CoA-SH) - Substrate level phosphorylation - Nucleoside diphosphate (NDP) kinase catalyzes GTP + ADP  GDP + ATP Synthetases vs synthase - Both types of enzymes are involved in joining substrates together - Synthase reactions do not require NTP (nucleotide triphosphates) - Synthetase reactions use NTP (ATP or GTP) as an energy source 21 Proposed mechanism of succinyl-CoA synthetase: 22 (6) Succinate dehydrogenase - a membrane-bound enzyme Q = ubiquinone QH2 = ubiquinol Malonate - A structural analog of succinate - Strong inhibitor of succinate dehydrogenase - Effectively blocks the TCA cycle activities 23 (7) Fumarase - Hydration of fumarate (8) Malate dehydrogenase 24 Fates of carbon atoms in the citric acid cycle: Oxaloacetate Succinate 25 ATP production from TCA cycle - Summary of TCA cycle: - Oxidation of reduced co-factors (NADH and QH2) in the electron transport chain (ETC) results in more ATP production 26 Net profit of aerobic degradation of glucose (32 ATP) 5 ATP (ETC = electron transport chain) 2 ATP 5 ATP (ETC) 15 ATP (ETC) QH2 3 ATP (ETC) 27 2 ATP Regulation of citric acid cycle - The cycle is precisely regulated to meet the cellular needs for ATP Pyruvate dehydrogenase NADH, acetyl CoA complex  NAD+, CoA-SH, pyruvate Citrate synthase ATP and NADH Isocitrate dehydrogenase NADH α-Ketoglutarate dehydrogenase NADH and succinyl CoA 28 TCA cycle is not always just a “cycle” The intermediates are involved in both catabolism and anabolism CR AR CR AR CR CR AR CR Cataplerotic reactions (CRs) AR - Depletion of citric acid cycle intermediates Anaplerotic reactions (ARs) AR AR - Filling up of citric acid cycle intermediates CR 29