TCA Cycle & Regulation PDF

Tricarboxylic Acid (TCA) Cycle Krebs cycle- Citric Acid Cycle- or Tricarboxylic Acid cyle (TCA) Key topics: – Learn the function of the pyruvate dehydrogenase complex, its reaction and control mechanisms – Learn the TCA sequence, enzymes, intermediates, products, and control mechanisms. – Understand...

Tricarboxylic Acid (TCA) Cycle Krebs cycle- Citric Acid Cycle- or Tricarboxylic Acid cyle (TCA) Key topics: – Learn the function of the pyruvate dehydrogenase complex, its reaction and control mechanisms – Learn the TCA sequence, enzymes, intermediates, products, and control mechanisms. – Understand the role of the cycle in providing biosynthetic precursors Cellular Respiration Captures energy Used by animals, plants, and many microorganisms Occurs in three major stages: - acetyl CoA production - acetyl CoA oxidation - electron transfer and oxidative phosphorylation Respiration: Stage 1 Acetyl-CoA Production Generates : ATP, NADH, FADH2 Respiration: Stage 2 Acetyl-CoA oxidation Generates more NADH, FADH2, and one GTP Respiration: Stage 3 Oxidative Phosphorylation In eukaryotes, citric acid cycle occurs in mitochondria Succinate dehydrogenase, is located in the inner membrane 1. Production of Acetyl-CoA Pyruvate must be converted to acetyl-CoA Pyruvate + coenzyme A + NAD+ → acetyl-CoA + CO2 + NADH Conversion of Pyruvate to Acetyl-CoA The Mitochondrial Pyruvate Carrier (MPC) Family) is embedded in the mitochondrial membrane. In the mitochondrial matrix --Oxidative decarboxylation of pyruvate Catalyzed by the pyruvate dehydrogenase complex (PDH) Pyruvate Dehydrogenase Complex (PDC) PDC is a large multienzyme complex - pyruvate dehydrogenase (E1) - dihydrolipoyl transacetylase (E2) - dihydrolipoyl dehydrogenase (E3) Requires 5 coenzymes – thiamine pyrophosphate, lipoic acid, and FAD are prosthetic groups – NAD+ and CoA-SH are co-substrates Arsenite : inhibits pyruvate dehydrogenase complex Advantages of multienzyme complexes: ‒ short distance between catalytic sites allows channeling of substrates from one catalytic site to another ‒ channeling minimizes side reactions ‒ regulation of activity of one subunit affects the entire complex Arsenite inhibits PDH complex throuh lipoic acid The Citric Acid Cycle (CAC) #1 Formation of Citrate #2 Isomerization by Dehydration/Rehydration Aconitase is an iron- sulfur protein Fluorocitrate : inhibits aconitase, causing citrate to accumulate # 3 Oxidation of Isocitrate to α-Ketoglutarate and CO2 Oxidative Decarboxylation Isocitrate Dehydrogenase #4 Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2 Oxidative Decarboxylation -Ketoglutarate Dehydrogenase - homologous to the pyruvate dehydrogenase complex thiamine pyrophosphate, lipoic acid, FAD, NAD+ and CoA-SH Active sites different to accommodate different-sized substrates Oxidative decarboxylation Succinyl-CoA : high-energy thioester molecule Arsenite inhibits the reaction Regulated by product inhibition #5 Conversion of Succinyl-CoA to Succinate Substrate level phosphorylation The enzyme also called SUCCINATE THIOKINASE – Gluconeogenic tissues (the liver and kidney): GDP izoenzyme – Nongluconeogenic tissues: ADP izoenzyme – connects TCA cycle to the other metabolic pathways – Can be synthesized from propionyl-coA #6 Oxidation of Succinate to Fumarate Succinate Dehydrogenase Iron- sulfur protein Bound to mitochondrial inner membrane – Part of Complex II in the electron-transport chain FAD : prosthetic group Malonate : competetive inhibitor #7 Hydration of Fumarate to Malate Fumarase catalyzes a stereospecific trans addition of H+ and OH- The OH- group adds to only one side of the double bond of fumarate; hence, only the L isomer of malate is formed. #8 Oxidation of Malate to Oxaloacetate Sequence of Events in the Citric Acid Cycle Step 1: C-C bond formation to make citrate Step 2: Isomerization via dehydration/rehydration Steps 3–4: Oxidative decarboxylations to give 2 NADH Step 5: Substrate-level phosphorylation to give GTP Step 6: Dehydrogenation to give FADH2 Step 7: Hydration Step 8: Dehydrogenation to give NADH Enzymes of the citric acid cycle may be physically associated with each other, leading products to pass directly from one to the other in a process called “substrate channeling” Citric acid cycle strictly aerobic because O2 required to regenerate NAD+ and FAD in the mitochondria One Turn of the Citric Acid Cycle Oxaloacetate is regenerated with each turn of the cycle The net reaction of the citric acid cycle is Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2H20 3NADH + FADH2 + GTP + 2H+ + CoA → 2 CO2 + Net Result of the Citric Acid Cycle Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O → 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+ Net oxidation of two carbons to CO2 – Equivalent to two carbons of acetyl-CoA Energy captured by electron transfer to NADH (and FADH2 Generates 1 GTP, which can be converted to ATP Completion of cycle Anaplerotic Reactions CAC intermediates are amphibolic Vitamins and minerals required for the TCA cycle and anaplerotic reactions – – – – – – – – – Niacin (NAD) (B3) Riboflavin (FAD) (B2) Pantothenatic Acid (CoA) Thiamine (B1) Biotin Mg Ca Fe Phosphate Regulation of the Citric Acid Cycle Regulated at irreversible steps – PDH, citrate synthase, Isocitrate DH, and α-k DH General regulatory mechanism – Activated by substrate availability – Inhibited by product accumulation – Overall products of the pathway are NADH and ATP Affect all regulated enzymes in the cycle Inhibitors: NADH and ATP Activators: NAD+ and AMP Regulation of the Citric Acid Cycle Regulation of Pyruvate Dehydrogenase The Pyruvate Dehydrogenase Complex is regulated allosterically and by reversible phosphorylation The complex also contains two tightly bound regulatory enzymes, pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. Regulation of Pyruvate Dehydrogenase By covalent protein modification: Rise in mitochondrial Ca2+ activates the phosphatase, enhancing pyruvate dehydrogenase activity. In tissues capable of fatty acid synthesis, such as the liver and adipose tissue: – In Adipose tissue: Insulin : stimulates the phosphatase the conversion of pyruvate into acetyl CoA – Acetyl CoA is the precursor for fatty acid synthesis. Inhibition of isocitrate dehydrogenase : buildup of citrate – Citrate -> transported to cytoplasm → signals glycolysis to stop – Can be a source of acetyl CoA for fatty acid synthesis Inhibition of α-ketoglutarate dehydrogenase – leads to buildup of α-ketoglutarate – used as precursor for synthesis of many amino acids and purine bases