Ch. 20 Tricarboxylic acid cyle Student Learning Outcomes: 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

TCA cycle (Kreb’s cycle) or citric acid cycle: Generates 2/3 of ATP 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

II. 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

A. Formation, oxidation of isocitrate: 2C onto oxaloacetate 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**

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**

D. Oxidation of Succinate to oxaloacetate: 2 e- from succinate 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**

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

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 succinate dehydrogenase: FAD transfers e- to Fe-S group and to ETC Fig. 4

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

Coenzymes CoASH, TPP (Figs. 8.11, 8.12)

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

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

Lipoate coenzyme: Lipoate is a 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

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

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

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

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

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

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

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

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

PDC regulated mostly by phosphorylation: Both enzymes in complex 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

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

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

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

TCA cycle accounts for about 2/3 of ATP generated from fuel oxidation 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

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

It is embedded in the inner mitochondrial membrane Review question Succinyl dehydrogenase 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