BISC 330L Lecture 30 Citric Acid Cycle pt 2 Chapter 17 Citrate has been made but the hydroxyl group is in the wrong place For the decarboxyla8ons that follow the OH group needs to be moved Citrate synthase reac8on mechanism Aconitase catalyzes a dehydra8on hydra8on reac8on to accomplish this Aconitase is an ironDsulfur protein Very common in oxDphos Clusters use iron Fe and inorganic sul de S to interact with cysteine sulfur atoms Cys In this example one Fe atom is bound to both COOD group and an OH group on citrate We now have a substrate ready for decarboxyla8on and capture of highDenergy electrons Conversion of isocitrate to D ketoglutarate generates NADH Citrate synthase reac8on mechanism Catalyzed by isocitrate dehydrogenase This reac8on generates NADH a carrier of highDtransferDpoten8al electron and this comes into play when we study how proton gradients generate ATP Another oxida8on decarboxyla8on reac8on Next Dketoglutarate is oxidized and decarboxylated to generate another NADH Another oxida8on decarboxyla8on reac8on Catalyzed by ketoglutarate dehydrogenase Mechanis8cally this reac8on is very similar to what we studied for the pyruvate dehydrogenase complex pyrvate CoA NAD Dketoglutarate CoA NAD succinyl CoA CO2 NADH acetyl CoA CO2 NADH Conversion of succinyl CoA to succinate generates GTP and thus ATP This 8me the highD energy thioester bond is used to drive phosphoryla8on of GDP to form GTP Conversion of succinyl CoA to succinate generates GTP and thus ATP Catalyzed by succinyl CoA synthetase This is the only CAC step that produces a compound with high phosphorylD transfer poten8al Cells can use the GTP as an energy source as it is readily converted to ATP GTP ADP GDP ATP catalyzed by nucleoside diphosphokinase Conversion of succinyl CoA to succinate rxn mechanism Catalyzed by succinyl CoA synthetase good example of energy transforma8on Step 1 orthophosphate reacts with succinyl CoA to form succinyl phosphate Step 2 ac8ve site his8dine then captures phosphoryl group and succinate released Step 3 Phosphohis8dine then swings away and interacts with GDP bound to ac8ve site genera8ng GTP and reseWng the enzyme G0 D3 4 kJ mol manifesta8on of par8cipa8on of highDenergy groups in all steps Succinyl CoA synthase 2 subunits subunit contains Rossman fold used to bind the ADP por8on of succinyl CoA subunit contain ATP grasp domain which in this case is used to bind GDP and ac8vate it for conversion to GTP Succinate serves as the founda8on for regenera8on of oxaloacetate Here we will see that a methylene group is converted to carbonyl group CH2 C O And along the way more highDenergy electrons are captured by NADH and FADH2 Succinate oxaloacetate succinate dehydrogenase 3 reac8ons oxida8on then hydra8on then oxida8on again The 1st rxn converts succinate to fumarate and generates FADH2 Rela8ve to NADH FADH2 is a minor contributor during oxida8ve phosphoryla8on as we ll see later FAD is the hydrogen acceptor in this rxn b c the freeDenergy change is not su cient to reduce NAD to NADH We will return to this rxn when we study oxDphos as it links the CAC to oxDphos Succinate oxaloacetate fumarase 3 reac8ons oxida8on then hydra8on then oxida8on again The 2nd rxn converts fumarate to malate by hydra8on Succinate oxaloacetate 3 reac8ons oxida8on then hydra8on then oxida8on again The 3rd rxn converts malate to oxaloacetate and resets the CAC This is the only rxn in the cycle with a large posi8ve freeDenergy change 29 7 kJ mol The rxn is driven to the right by u8liza8on of the rxn products NADH is consumed during oxDphos oxaloacetate is consumed by citrate synthase at beginning of CAC dehydrogenase malate Rxns of the CAC Summary 1 2 C atoms enter the cycle during condensa8on of acetyl CoA with oxaloacetate 2 C atoms leave in the form of CO2 2 4 pairs of H atoms leave the cycle in 4 oxida8on reac8ons to generate 3 NADH and 1 FADH2 Note that 1 NADH is also produced in genera8ng acetyl CoA from pyruvate 3 1 GTP is produced 4 2 molecules of water are consumed Rxns of the CAC Net reac8on Acetyl CoA 3 NAD FAD GDP Pi 2 H20 GTP 2 H CoA 2 CO2 3 NADH FADH2 Tracking the 2DC acetyl group that enters the CAC These carbons can be followed un8l the reac8on that converts succinyl CoA to succinate Note that the carbons that enter the cycle are not released during 1 turn DD these carbons can only be released in subsequent turns Regula8on of the CAC gluconeogenesis glycolysis irreversible rateDlimi8ng cri8cal branchDpoint for all of metabolism CAC lipid biogenesis metabolism Regula8on of pyruvate dehydrogenase PDH acetyl CoA CO2 NADH H Pyruvate CoA NAD PDH Regula8on of pyruvate dehydrogenase PDH Downstream outputs ac8vate PDH kinase and thereby turn PDH o Pyruvate and ADP inhibit the PDH kinase thereby promo8ng PDH ac8va8on Other CAC control points Isocitrate dehydrogenase is s8mulated by ADP allosterically which makes binding of isocitrate and NAD easier ATP inhibits isocitrate dehydrogenase as does NADH by displacing NAD from the enzyme Dketoglutarate dehydrogenase is also nega8vely regulated by both energy charge and its reac8on products succinyl CoA and NADH These control points regulate the rate of the CAC so that it is constantly in tune with the animal s need for ATP The CAC provides building blocks for numerous biosynthe8c pathways Ques8on if oxaloacetate is pulled from the cycle by a cell s demand for biosynthesis how is it replenished so that energy demand is also accommodated Oxaloacetate can be synthesized directly from pyruvate as we have seen during gluconeogenesis The glyoxylate cycle Opera8onal in some plants and microorganisms Allows these organisms to subside on acetate as sole source of fuel Bypasses the 2 decarboxyla8on steps in the CAC