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Chapt. 20 TCA cycle

Chapt. 20 TCA cycle. Ch. 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, CO 2 produced

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Chapt. 20 TCA cycle

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  1. Chapt. 20 TCA cycle • Ch. 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+ ratio

  2. Overview TCA cycle • TCA 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 GTP Fig. 1

  3. II. 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 group Fig. 2

  4. TCA cycle reactions • TCA 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 -> CO2 • also get NADH Fig. 3**

  5. TCA cycle reactions • TCA cycle Reactions. • B. a-ketoglutarate to Succinyl CoA: • Oxidative decarboxylation • releases CO2 • Succinyl joins to CoA • NADH formed • GTP made from • activated succinyl CoA Fig. 3**

  6. TCA cycle reactions • TCA cycle Reactions. • D. Oxidation of Succinate • to oxaloacetate: • 2 e- from succinate • to FAD-> FAD(2H) • Fumarate formed • H2O added -> malate • 2 e- to NAD+ -> NADH • Oxaloacetate restored • (common series of oxidations • to C=C, add H2O -> -OH, • oxidize -OH to C=O) Fig. 3**

  7. 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. 5

  8. III. Coenzymes are critical for TCA cycle • FAD can accept e- singly (as C=C formation) • FAD remains tightly bound to enzymes Fig. 6 membrane bound succinatedehydrogenase: FAD transfers e- to Fe-S group and to ETC Fig. 4

  9. Coenzyme CoA in TCA cycle • CoASH coenzyme forms thioester bond: • High energy bond • (Fig. 8.12 structure of CoASH formed from pantothenate) Fig. 7

  10. Coenzymes CoASH; TPP • Coenzymes CoASH, TPP • (Figs. 8.11, 8.12)

  11. Coenzymes in a-ketoacid dehydrogenase complex. • C. a-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 each Fig. 8

  12. a-ketoacid dehydrogenase enzyme complex: • 3 enzymes E1, E2, E3 • Coenzymes: TPP(thiamine pyrophosphate). • Lipoate, FAD Fig. 9

  13. Lipoate is a coenzyme • Lipoate 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 FAD Fig. 10

  14. Energetics of TCA cycle • Energetics of TCA cycle: overall net -DG0’ • Some reactions positive; • Some loss of energy as heat (-13 kcal) • Oxidation of NADH, • FAD(2H) helps pull • TCA cycle forward • Very efficient cycle: • Yield 207 Kcal from • 1 Acetyl -> CO2 • (90% theoretical 228) • Table 20.1 Fig. 11

  15. V. Regulation of TCA cycle • Many points of regulation of TCA cycle: • PO4 state of ATP (ATP:ADP) • Reduction state of NAD+ (ratio NADH:NAD+) • NADH must enter ETC Fig. 12

  16. Table 20.2 general regulatory mechanisms • Table 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 inhibitor

  17. Citrate synthase simple regulation • Citrate 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 citrate

  18. Allosteric regulation of isocitrate Dehydrogenase • Isocitrate dehydrogenase (ICDH): • Rate-limiting step • Allosteric activation by ADP • Small inc ADP -> large change rate • Allosteric inhibition by NADH • Reflect function of ETC Fig. 13

  19. Other regulation of TCA • Regulation of a-ketoglutarate dehydrogenase: • Product inhibited by NADH, succinyl CoA • May be inhibited by GTP • Like ICDH, responds to levels ADP, ETC activity • Regulation of TCA cycle intermediates: • Ensures NADH made fast enough for ATP homeostasis • Keeps concentration of intermediates appropriate

  20. VI. Precursors of Acetyl CoA • VI. Many fuels feed directly into Acetyl CoA • Will be completely oxidized to CO2 Fig. 14

  21. Pyruvate Dehydrogenase complex (PDC) • Pyruvate Dehydrogenase complex (PDC): • Critical step linking glycolysis to TCA • Similar to aKGDH (Fig. 20.15) • Huge complex; • Many copies each subunit: (Beef heart 30 E1, 60 E2, 6 E3, X) Fig. 15

  22. Regulation of PDC • PDC 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, NADH Fig. 16

  23. TCA cycle intermediates and anaplerotic paths • TCA cycle intermediates - biosynthesis precursors • Liver ‘open cycle’ high efflux of intermediates: • Specific transporters inner mitochondrial membrane for pyruvate, citrate, a-KG, malate, ADP, ATP. Fig. 17 GABA

  24. Anaplerotic reactions • Anaplerotic reactions replenish 4-C needed to regenerate oxaloacetate and keep TCA cycling: • Pyruvate carboxylase • Contains biotin • Forms intermediate with CO2 • Requires ATP, Mg2+ (Fig. 8.12) • Found in many tissues Fig. 18

  25. Amino acid degradation forms TCA cycle intermediates • Amino acid oxidation forms many TCA cycle intermediates: • Oxidation of • even-chain fatty acids and • ketone body not replenish Fig. 19

  26. Key concepts • TCA cycle accounts for about 2/3 of ATP generated from fuel oxidation • Enyzmes are all located in mitochondrial • Acetyl CoA is substrate for TCA cycle: • Generates CO2, NADH, FAD(2H), GTP • e- from NADH, FAD(2H) to electron-transport chain. • Enzymes need many cofactors • Intermediates of TCA cycle are used for biosynthesis, replaced by anaplerotic (refilling) reactions • TCA cycle enzymes are carefully regulated

  27. Nuclear-encoded proteins in mitochondria • Nuclear-encoded proteins enter mitochondria via translocases: • Proteins made on free ribosomes, bound with chaperones • N-terminal aa presequences • TOM complex crosses outer • TIM complex crosses inner • Final processing • Membrane proteins similar Fig. 20

  28. Review question • Succinyldehydrogenase differs from other enzymes in the TCA cycle in that it is the only enzyme that displays which of the following characteristics? • It is embedded in the inner mitochondrial membrane • It is inhibited by NADH • It contains bound FAD • It contains fe-S centers • It is regulated by a kinase

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