Chapt. 20 TCA cycleOverview TCA cycleII. Reactions of TCA cycleTCA cycle reactionsSlide 5Slide 6III. Coenzymes are critical: NAD+III. Coenzymes are critical for TCA cycleCoenzyme CoA in TCA cycleCoenzymes CoASH; TPPCoenzymes in a-ketoacid dehydrogenase complex.Slide 12Lipoate is a coenzymeEnergetics of TCA cycleV. Regulation of TCA cycleTable 20.2 general regulatory mechanismsCitrate synthase simple regulationAllosteric regulation of isocitrate DehydrogenaseOther regulation of TCAVI. Precursors of Acetyl CoAPyruvate Dehydrogenase complex (PDC)Regulation of PDCTCA cycle intermediates and anaplerotic pathsAnaplerotic reactionsAmino acid degradation forms TCA cycle intermediatesKey conceptsNuclear-encoded proteins in mitochondriaReview questionChapt. 20 TCA cycleCh. 20 Tricarboxylic acid cyle Student Learning Outcomes:• Describe relevance of TCA cycle•Acetyl CoA funnels products• Describe reactions of TCA cycle in cell respiration: 2C added, oxidations, rearrangements-> NADH, FAD(2H), GTP, CO2 produced• Explain TCA cycle intermediates are used in biosynthetic reactions• Describe how TCA cycle is regulated by ATP demand: ADP levels, NADH/NAD+ ratioOverview TCA cycleFig. 1TCA cycle (Kreb’s cycle) or citric acid cycle:•Generates 2/3 of ATP•2C unit Acetyl CoA•Adds to 4C oxaloacetate•Forms 6C citrate•Oxidations, rearrangements ->•Oxaloacetate again•2 CO2 released•3 NADH, 1 FAD(2H)•1 GTPII. Reactions of TCA cycle Reactions of TCA cycle:•2 C of Acetyl CoA are oxidized to CO2 (not the same 2 that enter)•Electrons conserved through NAD+, FAD -> go to electron transport chain•1 GTP substrate level phosphorylation:•2.5 ATP/NADH; 1.5 ATP/FAD(2H)• Net 10 high-energy P/Acetyl groupFig. 2TCA cycle reactionsTCA cycle Reactions.A. Formation, oxidation of isocitrate:2C onto oxaloacetate (synthase C-C synthetases need ~P)Aconitrase move OH(will become C=O)Isocitrate Dehydrogenase oxidizes –OH, cleaves COOH -> CO2also get NADH Fig. 3**TCA cycle reactionsTCA cycle Reactions.B. -ketoglutarate to Succinyl CoA:Oxidative decarboxylationreleases CO2Succinyl joins to CoANADH formedGTP made from activated succinyl CoA Fig. 3**TCA cycle reactionsTCA cycle Reactions.D. Oxidation of Succinate to oxaloacetate:2 e- from succinate to FAD-> FAD(2H)Fumarate formedH2O added -> malate2 e- to NAD+ -> NADHOxaloacetate restored(common series of oxidationsto C=C, add H2O -> -OH, oxidize -OH to C=O) Fig. 3**III. Coenzymes are critical: NAD+•Many dehydrogenases use NAD+ coenzyme•NAD+ accepts 2 e- (hydride ion H-): -OH -> C=O•NAD+, and NADH are released from enzyme;•Can bind and inhibit different dehydrogenases•NAD+/NADH regulatory role (e-transport rate)Fig. 5III. Coenzymes are critical for TCA cycle•FAD can accept e- singly (as C=C formation)•FAD remains tightly bound to enzymesFig. 4Fig. 6 membrane bound succinate dehydrogenase:FAD transfers e- to Fe-S group and to ETCCoenzyme CoA in TCA cycleCoASH coenzyme forms thioester bond:•High energy bond(Fig. 8.12 structure of CoASH formed from pantothenate)Fig. 7Coenzymes CoASH; TPPCoenzymes CoASH, TPP(Figs. 8.11, 8.12)Coenzymes in -ketoacid dehydrogenase complex.Fig. 8C. -ketoacid dehydrogenase complex: •3 member family (pyruvate dehydrogenase, branched-chain aa dehydrogenase)•Ketoacid is decarboxylated•CO2 released•Keto group activated, attached CoA•Huge enzyme complexes •(3 enzymes E1, E2, E3)•Different coenzymes in eachFig. 9-ketoacid dehydrogenase enzyme complex: •3 enzymes E1, E2, E3•Coenzymes: TPP(thiamine pyrophosphate). Lipoate, FADLipoate is a coenzymeLipoate coenzyme:•Made from carbohydrate, aa•Not from vitamin precursor•Attaches to –NH2 of lysine of enzyme•Transfers acyl fragment to CoASH•Transfers e- from SH to FADFig. 10Energetics of TCA cycleFig. 11Energetics of TCA cycle: overall net -G0’•Some reactions positive; •Some loss of energy as heat (-13 kcal)•Oxidation of NADH,FAD(2H) helps pull TCA cycle forwardVery efficient cycle:•Yield 207 Kcal from1 Acetyl -> CO2 •(90% theoretical 228)•Table 20.1V. Regulation of TCA cycleFig. 12Many points of regulation of TCA cycle:•PO4 state of ATP (ATP:ADP)•Reduction state of NAD+ (ratio NADH:NAD+)•NADH must enter ETCTable 20.2 general regulatory mechanismsTable 20.2 general regulation metabolic paths•Regulation matches function (tissue-specific differences)•Often at rate-limiting step, slowest step •Often first committed step of pathway, or branchpoint•Regulatory enzymes often catalyze physiological irreversible reactions (differ in catabolic, biosynthetic paths)•Often feedback regulation by end product•Compartmentalization also helps control access to enzymes•Hormonal regulation integrates responses among tissues:•Phosphorylation state of enyzmes•Amount of enzyme•Concentration of activator or inhibitorCitrate synthase simple regulationCitrate synthase simple regulation:•Concentration of oxaloacetate, the substrate•Citrate is product inhibitor, competitive with S•Malate -> oxoaloacetate favors malate•If NADH/NAD+ ratio decreases, more oxaloacetate•If isocitrate dehydrogenase activated, less citrateAllosteric regulation of isocitrate DehydrogenaseIsocitrate dehydrogenase (ICDH):•Rate-limiting step•Allosteric activation by ADP•Small inc ADP -> large change rate•Allosteric inhibition by NADH•Reflect function of ETCFig. 13Other regulation of TCARegulation of a-ketoglutarate dehydrogenase:•Product inhibited by NADH, succinyl CoA•May be inhibited by GTP•Like ICDH, responds to levels ADP, ETC activityRegulation of TCA cycle intermediates:•Ensures NADH made fast enough for ATP homeostasis•Keeps concentration of intermediates appropriateVI. Precursors of Acetyl CoAVI. Many fuels feed directly into Acetyl CoA•Will be completely oxidized to CO2 Fig. 14Pyruvate Dehydrogenase complex (PDC)Fig. 15Pyruvate Dehydrogenase complex (PDC):•Critical step linking glycolysis to TCA•Similar to KGDH (Fig. 20.15)•Huge complex; •Many copies each subunit:(Beef heart 30 E1, 60 E2, 6 E3, X)Regulation of PDCFig. 16PDC regulated mostly by phosphorylation:•Both enzymes in complex•PDC kinase add PO4 to ser on E1•PDC phosphatase removes PO4•PDC kinase:• inhibited by ADP, pyruvate•Activated by Ac CoA, NADHTCA cycle intermediates and anaplerotic pathsFig. 17GABATCA cycle intermediates - biosynthesis precursors•Liver ‘open cycle’ high efflux of