Bài giảng Biochemistry 2/e - Chapter 20: The Tricarboxylic Acid Cycle

Chapter 20The Tricarboxylic Acid Cycleto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Outline20.3 Bridging Step - Pyruvate Decarboxylase 20.4 Entry - Citrate Synthase 20.5 - 20.11 All the Other Steps 20.13 Intermediates for Other Pathways 20.14 Anaplerotic Reactions 20.15 Regulation of the TCA Cycle 20.16 The Glyoxylate CycleThe TCA Cycleaka Citric Acid Cycle, Krebs Cycle Pyruvate (actually acetate) from glycolysis is degraded to CO2 Some ATP is produced More NADH is made NADH goes on to make more ATP in electron transport and oxidative phosphorylationThe Chemical Logic of TCA Understand this! TCA seems like a complicated way to oxidize acetate units to CO2 But normal ways to cleave C-C bonds and oxidize don't work for CO2: cleavage between Cs  and  to a carbonyl an -cleavage of an  -hydroxyketone The Chemical Logic of TCA A better way to cleave acetate... Better to condense acetate with oxaloacetate and carry out a  -cleavage - TCA combines this with oxidation to form CO2, regenerate oxaloacetate and capture all the energy as NADH and ATP! Entry into the TCA CyclePyruvate dehydrogenase and citrate synthase Pyruvate is oxidatively decarboxylated to form acetyl-CoA Pyruvate dehydrogenase uses TPP, CoASH, lipoic acid, FAD and NAD+ Citrate synthase is classic CoA chemistry! Know both mechanisms NADH & succinyl-CoA are allosteric inhibitors Note (Table 20.1) that CS has large, neg G!AconitaseIsomerization of Citrate to Isocitrate Citrate is a poor substrate for oxidation So aconitase isomerizes citrate to yield isocitrate which has a secondary -OH, which can be oxidized Note the stereochemistry of the Rxn: aconitase removes the pro-R H of the pro-R arm of citrate! Aconitase uses an iron-sulfur cluster - see Fig. 20.8Isocitrate DehydrogenaseOxidative decarboxylation of isocitrate to yield  -ketoglutarate Classic NAD+ chemistry (hydride removal) followed by a decarboxylation Isocitrate dehydrogenase is a link to the electron transport pathway because it makes NADH Know the mechanism! -Ketoglutarate DehydrogenaseA second oxidative decarboxylation This enzyme is nearly identical to pyruvate dehydrogenase - structurally and mechanistically Five coenzymes used - TPP, CoASH, Lipoic acid, NAD+, FAD You know the mechanism if you remember pyruvate dehydrogenase Succinyl-CoA SynthetaseA substrate-level phosphorylation A nucleoside triphosphate is made Its synthesis is driven by hydrolysis of a CoA ester The mechanism (Figure 20.13) involves a phosphohistidine Succinate DehydrogenaseAn oxidation involving FAD Mechanism involves hydride removal by FAD and a deprotonation This enzyme is actually part of the electron transport pathway in the inner mitochondrial membrane The electrons transferred from succinate to FAD (to form FADH2) are passed directly to ubiquinone (UQ) in the electron transport pathwayFumaraseHydration across the double bond trans-addition of the elements of water across the double bond Possible mechanisms are shown in Figure 20.18 The actual mechanism is not known for certain Malate DehydrogenaseAn NAD+-dependent oxidation The carbon that gets oxidized is the one that received the -OH in the previous reaction This reaction is energetically expensive Go' = +30 kJ/mol This and the previous two reactions form a reaction triad that we will see over and over! TCA Cycle SummaryOne acetate through the cycle produces two CO2, one ATP, four reduced coenzymes Make sure that you understand the equations on page 659 A healthy exercise would be to try to derive these equations (or at least justify each term) The Fate of Carbon in TCAStudy Figure 20.21 carefully! Carboxyl C of acetate turns to CO2 only in the second turn of the cycle (following entry of acetate) Methyl C of acetate survives two cycles completely, but half of what's left exits the cycle on each turn after that. Intermediates for Biosynthesis The TCA cycle provides several of these  -Ketoglutarate is transaminated to make glutamate, which can be used to make purine nucleotides, Arg and Pro Succinyl-CoA can be used to make porphyrins Fumarate and oxaloacetate can be used to make several amino acids and also pyrimidine nucleotides Intermediates for Biosynthesis The TCA cycle provides several of these Note (Fig. 20.23) that mitochondrial citrate can be exported to be a cytoplasmic source of acetyl-CoA and oxaloacetateThe Anaplerotic ReactionsThe "filling up" reactions PEP carboxylase - converts PEP to oxaloacetate Pyruvate carboxylase - converts pyruvate to oxaloacetate Malic enzyme converts pyruvate into malate PEP carboxykinase - could have been an anaplerotic reaction, but it goes the wrong way! CO2 binds weakly to the enzyme, but oxaloacetate binds tightly, so the reaction goes the wrong way.The Reductive TCA CycleThe TCA cycle running backward could assimilate CO2This may have been the first metabolic pathwayEnergy to drive it? Maybe reaction of FeS with H2S to form FeS2 (iron pyrite) iron pyrite, which was plentiful in ancient times, and which is an ancient version of ‘iron-sulfur clusters’!Regulation of the TCA CycleAgain, 3 reactions are the key sites Citrate synthase - ATP, NADH and succinyl-CoA inhibit Isocitrate dehydrogenase - ATP inhibits, ADP and NAD+ activate  -Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit, AMP activates Also note pyruvate dehydrogenase: ATP, NADH, acetyl-CoA inhibit, NAD+, CoA activate The Glyoxylate CycleA variant of TCA for plants and bacteria Acetate-based growth - net synthesis of carbohydrates and other intermediates from acetate - is not possible with TCA Glyoxylate cycle offers a solution for plants and some bacteria and algae The CO2-evolving steps are bypassed and an extra acetate is utilized Isocitrate lyase and malate synthase are the short-circuiting enzymes Glyoxylate Cycle IIIsocitrate lyase produces glyoxylate and succinate Malate synthase does a Claisen condensation of acetyl-CoA and the aldehyde group of glyoxylate - classic CoA chemistry! The glyoxylate cycle helps plants grow in the dark! Glyoxysomes borrow three reactions from mitochondria: succinate to oxaloacetate