Citric Acid Cycle PDF

Citric Acid Cycle Hans Krebs (1953 Nobel Prize in Physiology/Medicine) and his cycle... also the citric acid cycle, tricarboxylic acid (TCA) cycle Citric Acid Cycle 1. Overview of the Hub 2. Acetyl-CoA & Pyruvate...

Citric Acid Cycle Hans Krebs (1953 Nobel Prize in Physiology/Medicine) and his cycle... also the citric acid cycle, tricarboxylic acid (TCA) cycle Citric Acid Cycle 1. Overview of the Hub 2. Acetyl-CoA & Pyruvate Dehydrogenase 3. Into the Cycle 4. Regulation 5. Metabolic Hub  Citric Acid Cycle 1. Overview of the Hub 2. Acetyl-CoA & Pyruvate Dehydrogenase 3. Into the Cycle 4. Regulation 5. Metabolic Hub 1. Overview & Orientation aerobic metabolism: further oxidation of pyruvate to CO2 and H 2O stage 1: generate acetyl-CoA stage 3: NADH & stage 2: FADH2 for acetyl groups oxidative into the citric phosphorylation acid cycle  1. Metabolic Hub for recovering energy from multiple metabolites The citric acid cycle is the central oxidative pathway in respiration, the process by which ALL metabolic fuels (carbohydrate, lipid, and protein) are catabolized in aerobic organisms and tissues. 1. Overall Input & Output + 3 NAD + FAD + GDP + Pi + acetyl-CoA 3 NADH + FADH2 + GTP + CoA + 2CO2 from 1 glucose to 2 pyruvate so, double the yields... 6 NADH + 2 FADH2 + 2 GTP + 2 CoA + 4 CO2  1. Localization in eukaryotes, all TCA cycle enzymes are in the mitochondrial matrix (oxidative phosphorylation occurs in the cristae) all substrates must be generated in or transported into the mitochondria in prokaryotes, all TCA cycle reactions occur in the cytosol remember: glycolysis is cytosolic in both prokaryotes and eukaryotes 1. Localization NOTE: The acetyl-CoA transport system is much more complicated than suggested here.  1. Pyruvate Symporter Pyruvate is taken up by a specific, low Km transporter that brings pyruvate into the mitochondrial matrix for oxidation by the pyruvate dehydrogenase complex. Citric Acid Cycle 1. Overview of the Hub 2. Acetyl-CoA & Pyruvate Dehydrogenase 3. Into the Cycle 4. Regulation 5. Metabolic Hub  2. Acetyl-CoA Synthesis connecting glycolysis and the citric acid cycle 2. Acetyl-CoA Synthesis most organisms can generate glucose from pyruvate animals cannot synthesize glucose from acetyl-CoA conversion of pyruvate to acetyl- CoA commits carbon to TCA cycle 2. Pyruvate Dehydrogenase Reaction To connect glycolysis with the citric acid cycle, the overall reaction is... + pyruvate + CoA + NAD acetyl-CoA + CO2 + NADH ie., oxidation of pyruvate (C3) to acetyl-CoA & CO2 2. Pyruvate Dehydrogenase Complex (PDC)  2. Pyruvate Dehydrogenase Complex (PDC) macromolecular complexes Signal Transduction/ Primary & Specialized Cell Cycle Translation/Transcription Metabolism Ubiquitin Ligase The Ribosome Fatty Acid & Polyketide Complex Synthases Cellular Structure Arp2/3 Complex What are the advantages? 2. Pyruvate Dehydrogenase Complex (PDC) What are the advantages? 1) Proximity of active sites maximizes reaction rates (think diffusion and collisions) 2) channeling of unstable/reactive metabolites (molecules never leave the enzyme or at least don’t go far away before hitting next active site) 3) coordinated regulation of multiple steps in a pathway (physical localization)  2. PDC Composition & Size Three components (in multiple copies) E1 - pyruvate dehydrogenase E2 - dihydrolipoyl transacetylase E3 - dihydrolipoyl dehydrogenase E. coli PDC complex 4,600 kDa - 300 Å in diameter (0.3 µm; think about glycogen granule size 0.1-0.4 µm) core of 24 E2 proteins surrounded by 24 E1 proteins and 12 E3 proteins In mammals, yeast, some bacteria, an even larger PDC 10,000 kDa (60 E2, 45 E1, 9 E3 plus others) NOTE: ribosomes are 3,000 to 6,000 kDa 2. Molecular Weight and Size 12 kDa 17 kDa 23 kDa 49 kDa 300 kDa 150 kDa 130 kDa  2. Molecular Weight and Size ribosome PDC 2. Organization of the PDC 24 E2 24 E1 24 E2 12 E3 24 E1 E1 - pyruvate dehydrogenase 12 E3 E2 - dihydrolipoyl transacetylase E3 - dihydrolipoyl dehydrogenase  2. PDC: Cryo-Electron Microscopy 122 cameras arranged in a circle to record images, then reconstruct the shot 2. PDC: Cryo-Electron Microscopy Image Processing Individual particles are Orientation of each ‘boxed’ and collected particle is calculated Milne et al (2002) EMBO J. 21, 5587  2. PDC: Cryo-Electron Microscopy Image Processing Fourier transform reassembles individual images that are assembled into a 3D image Milne et al (2002) EMBO J. 21, 5587 2. PDC: Cryo-Electron Microscopy Fit known structures into the model E2 catalytic subunits E1 tetramers Milne et al (2002) EMBO J. 21, 5587  2. PDC: Cryo-Electron Microscopy modeled lipoyl arms E2 catalytic subunits E1 tetramers Milne et al (2002) EMBO J. 21, 5587 2. Eukaryote PDC core: Cryo-EM only the E2 catalytic subunit assembly at 8.8 Å resolution Yu et al (2008) Structure 16, 104  2. Pyruvate Dehydrogenase Reaction To connect glycolysis with the citric acid cycle, the overall reaction is... + pyruvate + CoA + NAD acetyl-CoA + CO2 + NADH oxidation of pyruvate to acetyl-CoA & CO2 2. PDC Reactions requires five distinct reactions and five different coenyzymes across E1, E2, and E3 E1 E3 E2 TPP = thiamine pyrophosphate FAD = flavin dinucleotide  2. PDC Cofactors 2. PDC Reactions requires five distinct reactions and five different coenyzymes across E1, E2, and E3 E1 E3 E2 TPP = thiamine pyrophosphate FAD = flavin dinucleotide  2. PDC Reactions - E1 1. E1: decarboxylation of pyruvate via thiamine pyrophosphate (TPP) 2. PDC Reactions - E2 requires five distinct reactions and five different coenyzymes across E1, E2, and E3 E1 E3 E2 oxidized reduced TPP = thiamine pyrophosphate FAD = flavin dinucleotide  2. PDC Reactions - E2 2. E2: transfer of hydroxyethyl from E1 to E2 the "swinging arms" of lipoic acid oxidized reduced 2. PDC Reactions - E2 2. E2: the "swinging arms" of lipoic acid  2. Aside: Arsenic Poisoning Arsenic poisoning (intentional and unintentional) has had a long history, dating back to at least the eighth century. Trivalent As(III) compounds such as arsenite and organic arsenicals react readily with thiols, and they are especially reactive with dithiols, such as dihydrolipoamide, forming bidentate adducts: 2. PDC Reactions - E2 2. E2: the "swinging arms" of lipoic acid modeled lipoyl arms E2 catalytic subunits E1 tetramers Milne et al (2002) EMBO J. 21, 5587  2. PDC Reactions - E2 2. E2: transfer of hydroxyethyl to E2 TPP TPP TPP 2. PDC Reactions - E2 requires five distinct reactions and five different coenyzymes across E1, E2, and E3 E1 E3 E2 oxidized reduced TPP = thiamine pyrophosphate FAD = flavin dinucleotide  2. PDC Reactions - E2 Coenyzyme A Free Thiol: A Handle for Chemical Attachment 2. PDC Reactions - E2 3. E2: acetylation of CoA "a thiol exchange” reaction reduced  2. PDC Reactions - E3 requires five distinct reactions and five different coenyzymes across E1, E2, and E3 E1 reduced E3 E2 oxidized oxidized reduced TPP = thiamine pyrophosphate FAD = flavin dinucleotide 2. PDC Reactions - E3 4. E3: regeneration of lipoamide reduced oxidized disulfide  2. PDC Reactions - E3 the active site of E3 - dihydrolipoamide dehydrogenase 2. PDC Reactions - E3 4. E3: reoxidation of E3 by NAD oxidized reduced oxidized reduced oxidized reduced oxidized  Citric Acid Cycle 1. Overview of the Hub 2. Acetyl-CoA & Pyruvate Dehydrogenase 3. Into the Cycle 4. Regulation 5. Metabolic Hub 3. The Citric Acid Cycle pyruvate symporter  3. Chemical Logic of the Cycle Goal: oxidize acetate units to CO2 this requires cleavage of CO-C connections in biological systems, this occurs at Cα-Cβ relative to a carbonyl problem: acetate (CH3-COOH) does not have a Cβ could hydroxylate - but this is energetically unfavorable solution: condense acetate to oxaloacetate, then perform the β-cleavage & combine with oxidation to form CO2, regenerate oxaloacetate, and capture the energy in NADH & ATP 3. Chemical Logic of the Cycle 1 reaction to link (and oxidize - 1C) 4 reactions to regenerate oxaloacetate 4 reactions to oxidize “pyruvate” - 2C  3. Chemical Logic of the Cycle 4 reactions to oxidize “pyruvate” - 2C 3. Introduce Two Carbons (Acetyl-CoA) Step 1. Citrate Synthase condenses the last product of the cycle with the product of the PDC... oxaloacetate (C4) + acetyl-CoA citrate (C6) + CoA open form closed form 3. Introduce Two Carbons (Acetyl-CoA) Step 1. Citrate Synthase acid-base chemistry all modifications occur on acetyl-CoA acid (donates proton) regenerate active site stabilization hydrolysis enolate base (abstracts proton) 3. Isomerize Citrate Step 2. Aconitase isomerization of citrate to isocitrate requires a [4Fe-4S] iron-sulfur cluster to facilitate elimination and addition of water tertiary to secondary alcohol (easier to oxidize)  3. Aside: Fe-S Clusters sulfide-linked multi-iron centers in different oxidation states metalloproteins - electron transport system 2Fe-2S cluster - coordinated by 4 ligands (4 cys or 2 cys/2his) oxidized Fe3+ Fe3+ reduced Fe3+ Fe2+ 3. Aside: Fe-S Clusters 4Fe-4S cluster - cubane-type architecture coordinated by cysteines In aconitase, isocitrate binds at the Fe lacking a ligand and the cluster does not change in redox state (i.e., not an electron carrier).  3. Isomerize Citrate Step 2. Aconitase isomerization of citrate to isocitrate [4Fe-4S] iron-sulfur cluster - 3 cysteines and 1 open site substrate binding and removal/addition of water from substrate 3. Generate First CO2 Step 3. Isocitrate Dehydrogenase isocitrate (C6) to α-ketoglutarate (C5) - oxidative decarboxylation α β α β α α enolate metal assisted catalysis Mn/Mg - polarize the carbonyl first NADH generated in cycle first CO2 generated in cycle  3. Generate CO2 Step 3. Isocitrate Dehydrogenase isocitrate (C6) to α-ketoglutarate (C5) - oxidative decarboxylation α β α β α enolate Oxidation of isocitrate (alcohol) oxalosuccinate (ketone) intermediate Oxalosuccinate is an unstable enzyme-bound intermediate that spontaneously β-decarboxylates to the product α-ketoglutarate. Strategy: oxidize the secondary alcohol (isocitrate) to a keto group at the β- positition to the carboxyl group to be removed (oxalosuccinate). The β-keto group acts as an electron sink to stabilize the carbanionic transition state, which facilitates decarboxylation. 3. Generate CO2 Step 3. Isocitrate Dehydrogenase two forms of this enzyme one uses NAD(H) & is found in the mitochondrial matrix (TCA cycle) the other uses NADP(H) & is localized to the mitochondrial matrix and the cytosol (other metabolism)  3. Generate the next CO2 Step 4. α-Ketoglutarate Dehydrogenase α-ketoglutarate (C5) to succinyl(C4)-CoA - oxidative decarboxylation second NADH from cycle & second CO2 released β β α α Does this reaction look familiar? Identical chemistry to pyruvate dehydrogenase! 3. Generate the next CO2 swap α-ketoglutarate for pyruvate and succinyl-CoA for acetyl-CoA the PDC and KDC have the same cofactors and architecture ketoglutarate TPP = thiamine pyrophosphate FAD = flavin dinucleotide  3. The Chemical Logic of the Cycle 4 reactions to regenerate oxaloacetate 3. Substrate-level Phosphorylation Step 5. Succinyl-CoA Synthetase succinyl-CoA + GDP succinate + GTP consists of two subunits: α: catalytic histidine and the CoA binding site β: nucleotide binding (GTP & ATP specific isoforms)  3. Substrate-level Phosphorylastion Step 5. Succinyl-CoA Synthetase Three successive nucleophilic substitution reactions conserve the energy of the thioester of succinyl-CoA in the phosphoanhydride bond of GTP another theme phosphorylation to generate a reactive intermediate phosphohistidine! 3. Flavin-dependent Dehydrogenation Step 6. Succinate Dehydrogenase Start of converting succinate (4C) to oxaloacetate (4C) FADH2 is a reducing equivalent for oxidative phosphorylation, but unlike NADH is not free to diffuse A single C-C bond is more difficult to oxidize than a C-O bond - need the more powerful oxidant FAD for this reaction  3. Flavin-dependent Dehydrogenation Step 6. Succinate Dehydrogenase eukaryotes: bound to the mitochondrial inner membrane prokaryotes: bound to the plasma membrane 3. Flavin-dependent Dehydrogenation Step 6. Succinate Dehydrogenase in addition to the FAD, there are three iron-sulfur clusters electrons pass from succinate to FAD to each Fe S cluster before entering the electron transport chain of oxidative phosphorylation  3. Hydrate the Double Bond Step 7. Fumarase: fumarate to malate 3. Dehydrate to Oxaloacetate Step 8. Malate Dehydrogenase third NADH from cycle MDH equilibrium favors malate not oxaloacetate but … coupling to citrate synthase (start of TCA cycle) draws the reaction forward  3. TCA Cycle: Carbon Flow 2C 4C 6C 4C 6C 4C 5C 4C 4C 3. TCA Cycle: Energy Generation  3. TCA Cycle: aerobic vs. anaerobic Ox-Phos homolactate fermentation 2 ATP Citric Acid Cycle 1. Overview of the Hub 2. Acetyl-CoA & Pyruvate Dehydrogenase 3. Into the Cycle 4. Regulation 5. Metabolic Hub  4. Cycle Regulation: First Bottleneck 1. PDC allosterically inhibited by ATP, NADH & acetyl-CoA & activated by insulin, ADP, pyruvate 2. Phosphorylation of E1 in the PDC (eukaryotes) 4. Cycle Regulation: First Bottleneck Phosphorylation of E1 in the PDC (eukaryotes) PD kinase is part of the PDC complex in eukaryotes! PD kinase - activated by NADH & acetyl-CoA to phosphorylate a serine to block decarboxylation of pyruvate (E1)  4. Cycle Regulation: First Bottleneck Phosphorylation of E1 in the PDC (eukaryotes) insulin - activates the phosphatase NADH & acetyl-CoA (activates PDC) activate the kinase (inactivate PDC) think about this in connection to glycolysis 4. Cycle Regulation: 3 Control Points  4. Cycle Regulation: 3 Control Points Cycle Regulation at the Three Exergonic Steps 4. Cycle Regulation: 3 Control Points Key molecules oxaloacetate concentration (substrate availability) acetyl-CoA feedback inhibition NADH feedback inhibition is the major control! relative intramitochondrial concentrations of NAD+ vs. NADH citrate synthase isocitrate dehydrogenase Calcium signal stimulates muscle contraction ketoglutarate (ADP signals need for fuel) dehydrogenase  Citric Acid Cycle 1. Overview of the Hub 2. Acetyl-CoA & Pyruvate Dehydrogenase 3. Into the Cycle 4. Regulation 5. Metabolic Hub 5. Metabolic Hub  5. Metabolic Hub malate & the oxaloacetate transport system any intermediate can be used for gluconeogenesis 5. Metabolic Hub PDC generates acetyl-CoA in mitochondria (not transported out) Fatty acid synthesis in the cytosol relies on acetyl- CoA, so... citrate (which is transportable) can be broken down to generate acetyl-CoA for FA  5. Metabolic Hub ketoglutarate & oxaloacetate are substrates for transamination reactions in amino acid synthesis 5. Metabolic Hub building block for porphyrins (hemes, chlorophyll) - needed for ox-phos & photosynthesis  5. Metabolic Hub ketoglutarate & oxaloacetate are also common amino acid breakdown products 5. Metabolic Hub specialized amino acid breakdown products - fumarate & succinyl-CoA  Next Semester … translation