Citric Acid Cycle 2024 (PDF)

The Citric Acid Cycle Marc A. Ilies, Ph. D. Lehninger - Chapter 16 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas...

The Citric Acid Cycle Marc A. Ilies, Ph. D. Lehninger - Chapter 16 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas ©2024MAIlies 1  Cellular respiration and energy production - aerobic phase of catabolism comprising processes by which cells consume O2 and produce CO2 and energy = cellular respiration - comprises three stages: 1. oxidation of organic fuels to acetylCoA 2. conversion of acetylCoA to CO2 and reduced forms of electron carriers NADH, FADH2 3. oxidation of electron carriers with O2 (final electron acceptor) with release of energy, converted to ATP (oxidative phosphorylation) 2  Cellular respiration and energy production - carried out in mitochondria: 3  Oxidation of pyruvate to acetylCoA - oxidative decarboxylation catalyzed by a cluster of enzymes and their cofactors = pyruvate dehydrogenase complex (PDH): It is an irreversible process !! - PDH complex essentially converts pyruvate (3C) to acetyl Co-A (2C), yielding CO2 and NADH; it has: 3 Enzymes 5 Cofactors: Thiamine Pyrophosphate (TPP), Lipoic Acid, NAD+, FAD, Coenzyme A (CoA-SH) (note that four different vitamins are involved: thiamine (B 1), riboflavin (B2), niacin (B3), and pantothenate (B5)) 4  Coenzyme A and acetylCoA - structure: 3’-phosphoadenosinediphosphate + pantothenic acid + mercaptoethanolamine: (pantothenic acid: vitamin B5) - acetylCoA is more energy-rich than ATP: 5  Lipoic acid: key cofactor - Lipoic acid (acting attached on Lys sidechain of dihydrolipoyl transacetylase) can undergo redox reactions and can attach acetyl groups (similar to CoA): 6  Oxidation of pyruvate to acetylCoA - five consecutive steps, achieved in PDH complex: (also called pyruvate decarboxylase) - PDH complex can be covalently modified by phosphorylation which makes it less active and dephosphorylation to make it more active. These processes are catalyzed by PDH- kinase and PDH-phosphatase. An inherited decreased activity of PDH results in Chronic Lactic Acidosis (CLA). 7  Oxidation of pyruvate to acetylCoA - PDH complex: supramolecular enzymatic complex of nanometric dimensions containing: E1 pyruvate dehydrogenase (aka pyruvate decarboxylase) E2 dihydrolipoyl transacetylase (20 trimers = 60 molecules) E3 dihydrolipoyl dehydrogenase - the swinging lipoyl arm of E2 (blue) can reach the active sites of E1 and E3 (enzymes are mechanically coupled!!!): 8  Step 1: Decarboxylation of pyruvate - achieved using TPP (vit B1) as coenzyme, which carries the product = acetaldehyde: Thiamine pyrophosphate O O E1 =decarboxylase H3C NH2 O P O P O E1 N E2 =transacetylase N O O S H3C N H TPP Electron "Sink" CO2 Hydroxyethyl TPP H3C E1 H3C E1 E1 H3C E1 H3C R N S N S S S R R N R N O H3C C C C H3C C OH O O H3C OH H3C C C OH H Enz-Base O O O S S Pyruvate E2 Lys HN C (CH2)4 (see also the fate of pyruvate in glycolysis) 9  Step 2: Oxidation of activated acetaldehyde to acetate - the “active” acetaldehyde bound on TPP is then transferred to the lipoyl moiety of dihydrolipoyl transacetylase (E2), where it undergoes an internal redox reaction: (contd) Free thiamine released to accept another pyruvate H3C E1 SCoA HS.. CoA H3C C O R N S O S SH H3C C O H3C C OH E2 Lys HN C (CH2)4 O S SH O S SH E2 Lys HN C (CH2)4 E2 Lys HN C (CH2)4 + 10  Steps 3,4: Reoxidation of reduced lipoyl group; e- harvesting - the reduced form of lipoic acid residue is oxidized to regenerate the active (disulfide) form: + FAD - FADH2 Dihydrolipoyl (active) dehydrogenase (E3) - FADH2 is converted to NADH (final acceptor of electrons harvested from pyruvate): FADH2 + NAD+ FAD + NADH + H + Dihydrolipoyl dehydrogenase (E3) Overall reaction (4 steps combined): 11  2C 4C 6C 4C 6C Citric acid cycle 6C 4C 4C 4C 5C 12  Citric acid cycle: steps - acetyl-CoA donates the acetyl group to oxaloacetate (4C) to form citrate (6C): (CoA-SH is recycled into oxidative decarboxylation of another pyruvate molecule) - citrate is isomerized to isocitrate by aconitase : 13  Citric acid cycle: steps - isocitrate (6C) undergoes oxidative decarboxylation to -ketoglutarate (5C) with simultaneous energy harvesting (NAD(P)H): - -ketoglutarate (5C) undergoes oxidative decarboxylation to succinate (4C) with simultaneous energy harvesting (NADH) and coupling with CoA-SH: Analogous to PDH Complex 14  Citric acid cycle: steps - energy stored in succinyl-CoA is transferred to GTP (CoA-SH recycled): - all following steps done to convert succinate to oxaloacetate in order to close the cycle; first succinate is oxidized to fumarate: Membrane Bound 15  Citric acid cycle: steps - fumarate is hydrated to malate: - malate is oxidized to oxaloacetate, closing the citric acid cycle: Overall: CH3CO-S-CoA + GDP + P + H2O + 3 NAD+ + FAD 2 CO2 + HS-CoA + GTP + 3 NADH + FADH2 16  6 6 6 6 4 6 38 We will use 3 ATP for each NADH and 2 ATP for each FADH2 17  TCA is amphibolic: anabolic and catabolic - citric acid components are intermediates in catabolic degradation of pyruvate but are also important biosynthetic intermediates: - shown in red are anaplerotic reactions 18 Anaplerotic reactions replenish citric acid cycle intermediates (anaplerotic = to fill up (gr.) (concentration of various intermediates in citric acid cycle is kept ~ constant through anaplerotic reactions) 19  Biological Tethers - carry 1 or 2 carbon intermediates from one active site to another: 20 Regulation of TCA and metabolic flow - regulation at the level of highly exothermic (essentially irreversible) reactions: 21 Fate of Citrate Citrate Shuttle 22 Sources and Fate of Succinate Heme 23  Goals and Objectives Upon completion of this series of lectures at minimum you should be able to answer the following: ► What is cellular respiration, what stages it comprises, which are the main intermediates and enzymes involved? Which is the ultimate electron acceptor in vivo, and which compounds are used in vivo for energy storage? ►Which are the main structural particularities of mitochondria and where are various enzymes relevant for cellular respiration placed? ►Which are the main steps, intermediates, and enzyme/enzyme complexes involved in oxidation of pyruvate to acetylCoA, what are their particularities? ►Which are the main steps, intermediates, and enzymes involved in the citric acid cycle, what are their particularities, how is it regulated, and by which chemical entities, what decides the fate of citrate, piruvate, succinate ? ►At which steps in the conversion of molecule of glucose through glycolysis, coupled with cellular respiration we consume/produce energy and what is the overall balance? ► Why we state that citric acid cycle is amphibolic, what are anaplerotic reactions (with examples), what is their significance? ►How is the citric acid coupled with other important physiologic processes? 24  Drugs and Diseases ►Drugs: thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), and pantothenic acid (vitamin B5)), biotin (vitamin B7) ►Dietary supplements: lipoic acid ►Diseases: Beri-Beri, ariboflavinosis, pellagra, chronic lactic acidosis (CLA) 25