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Chapter 19

Chapter 19. The Tricarboxylic Acid Cycle Biochemistry by Reginald Garrett and Charles Grisham. Essential Question. How is pyruvate oxidized under aerobic conditions Under aerobic conditions, pyruvate is converted to acetyl-CoA and oxidized to CO 2 in the TCA cycle

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Chapter 19

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  1. Chapter 19 The Tricarboxylic Acid Cycle Biochemistry by Reginald Garrett and Charles Grisham

  2. Essential Question • How is pyruvate oxidized under aerobic conditions Under aerobic conditions, pyruvate is converted to acetyl-CoA and oxidized to CO2 in the TCA cycle • What is the chemical logic that dictates how this process occurs?

  3. Figure 19.1 (a) Pyruvate produced in glycolysis is oxidized in (b) the tricarboxylic acid (TCA) cycle. (c) Electrons liberated in this oxidation flow through the electron-transport chain and drive the synthesis of ATP in oxidative phosphorylation. In eukaryotic cells, this overall process occurs in mitochondria.

  4. Hans Krebs showed that the oxidation of acetate is accomplished by a cycle TCA cycle, Citric Acid Cycle or Krebs Cycle • Pyruvate from glycolysis is oxidatively decarboxylated to acetate and then degraded to CO2 in TCA cycle • Some ATP is produced • More NADH and FADH2are made • NADH goes on to make more ATP in electron transport and oxidative phosphorylation (chapter20)

  5. Figure 19.2The tricarboxylic acid cycle.

  6. 19.1 – What Is the Chemical Logic of the TCA Cycle? • TCA cycle 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 acetyl-CoA: • cleavage between Carbons  and to a carbonyl group (aldolase) • -cleavage of an -hydroxyketone (transaldolase) O —C—Ca— Cb— C OH —C—Ca—

  7. The Chemical Logic of TCAA better way to cleave acetate... • Better to condense acetate with oxaloacetate and carry out a-cleavage. • TCA combines this -cleavagereaction with oxidation to form CO2, regenerate oxaloacetate and capture all the energy in NADH and ATP

  8. 19.2 – How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA? • Pyruvate must enter the mitochondria to enter the TCA cycle • Oxidative decarboxylation of pyruvate is catalyzed by the pyruvate dehyrogenase complex Pyruvate + CoA + NAD+→ acetyl-CoA + CO2 + NADH + H+ • Pyruvate dehydrogenase complex is a noncovalent assembly of three enzymes • Five coenzymes are required

  9. Pyruvate dehydrogenase complex: Three enzymes and five coenzymes E1: pyruvate dehydrogenase (24) thiamine pyrophosphate E2: dihydrolipoyl transacetylase (24) lipoic acid E3: dihydrolipoyl dehydrogenase (12) FAD NAD+ CoA

  10. Pyruvate Dehydrogenase is a Multi-Subunit Complex of Three Enzymes Figure 19.3 Icosahedral model of the pyruvate dehydrogenase complex (PDC) core structure. E1 subunits are yellow; E2 subunits are green. Linkers in blue; E3 not shown.

  11. Figure 19.4 The Reaction Mechanism of the Pyruvate Dehydrogenase Complex

  12. Figure 19.5 The mechanism of the first three steps of the pyruvate dehydrogenase complex reaction

  13. The Coenzymes of the Pyruvate Dehydrogenase Complex Thiamine pyrophosphate TPP assists in the decarboxylation of α-keto acids (here) and in the formation and cleavage of α-hydroxy ketones (as in the transketolase reaction; see Chapter 22).

  14. The Coenzymes of the Pyruvate Dehydrogenase Complex The Nicotinamide Coenzymes – NAD+/NADH and NADP+/NADPH carry out hydride (H:-) transfer reactions. All reactions involving these coenzymes are two-electron transfers.

  15. The Coenzymes of the Pyruvate Dehydrogenase Complex The Flavin Coenzymes – FAD/FADH2 Flavin coenzymes can exist in any of three oxidation states, and this allows flavin coenzymes to participate in one-electron and two-electron transfer reactions. Partly because of this, flavoproteins catalyze many reactions in biological systems and work with many electron donors and acceptors.

  16. The Coenzymes of the Pyruvate Dehydrogenase Complex • Coenzyme A • The two main functions of Co A are: • Activation of acyl groups for transfer by nucleophilic attack • Activation of the α-hydrogen of the acyl group for abstraction as a proton The reactive sulfhydryl group on CoA mediates both of these functions. The sulfhydryl group forms thioester linkages with acyl groups. The two main functions of CoA are illustrated in the citrate synthase reaction (see Figure 19.6).

  17. The Coenzymes of the Pyruvate Dehydrogenase Complex Lipoic Acid functions to couple acyl-group transfer and electron transfer during oxidation and decarboxylation of α-keto acids. It is found in pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Lipoic acid is covalently bound to relevant enzymes through amide bond formation with the ε-NH2 group of a lysine side chain.

  18. 19.3 – How Are Two CO2 Molecules Produced from Acetyl-CoA? Tricarboxylic acid cycle, Citric acid cycle, and Krebs cycle • Pyruvate is oxidatively decarboxylated to form acetyl-CoA Citrate (6C)→ Isocitrate (6C)→a-Ketoglutarate (5C) →Succinyl-CoA (4C) → Succinate (4C) → Fumarate (4C) → Malate (4C) → Oxaloacetate(4C)

  19. Figure 19.2The tricarboxylic acid cycle.

  20. Citrate synthase reaction • Perkin condensation: a carbon-carbon condensation between a ketone or aldehyde and an ester Figure 19.6 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA. The mechanism involves nucleophilic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis.

  21. Citrate synthase • is a dimer • NADH & succinyl-CoA are allosteric inhibitors • Large, negative G -- irreversible Figure 19.7 Citrate synthase in mammals is a dimer of 49-kD subunits. In the monomer shown here, citrate (blue) and CoA (red) bind to the active site, which lies in a cleft between two domains and is surrounded mainly by α-helical segments.

  22. Citrate Is Isomerized by Aconitase to Form Isocitrate Isomerization 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 reaction: aconitase removes the pro-R H of the pro-R arm of citrate • Aconitase uses an iron-sulfur cluster ( Fig. 19.8)

  23. Figure 19.8 (a) The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-R hydrogen from the pro-R arm of citrate. (b) The active site of aconitase. The iron-sulfur cluster (pink) is coordinated by cysteines (orange) and isocitrate (purple).

  24. Aconitase Utilizes an Iron-Sulfur Cluster Figure 19.9 The iron-sulfur cluster of aconitase. Binding of Fe2+ to the vacant position of the cluster activates aconitase. The added iron atom coordinates the C-3 carboxyl and hydroxyl groups of citrate and acts as a Lewis acid, accepting an electron pair from the hydroxyl group and making it a better leaving group.

  25. Fluoroacetate is an extremely poisonous agent that blocks the TCA cycle • Rodent poison: LD50 is 0.2 mg/kg body weight • Aconitase inhibitor

  26. Isocitrate Dehydrogenase Catalyzes the First Oxidative Decarboxylation in the Cycle Oxidative decarboxylation of isocitrate to yield -ketoglutarate • Catalyzes the first oxidative decarboxylation in the cycle • Oxidation of C-2 alcohol of isocitrate with concomitant reduction of NAD+ to NADH • followed by a b-decarboxylation reaction that expels the central carboxyl group as CO2

  27. Isocitrate Dehydrogenase • Isocitrate dehydrogenase is a link to the electron transport pathway because it makes NADH • Isocitrate dehydrogenase is a regulation reaction • NADH and ATP are allosteric inhibitor • ADP acts as an allosteric activator • -ketoglutarate is also a crucial a-keto acid for aminotransferase reactions (Chapter 25), connecting the TCA cycle with nitrogen metabolism

  28. Figure 19.10(a) The isocitrate dehydrogenase reaction. (b) The active site of isocitrate dehydrogenase. Isocitrate is shown in green, NADP+ is shown in gold, with Ca2+ in red.

  29. -Ketoglutarate Dehydrogenase • Catalyzes the second oxidative decarboxylation of the TCA cycle • This enzyme is nearly identical to pyruvate dehydrogenase - structurally and mechanistically • a-ketoglutarate dehydrogenase • Dihydrolipoyl transsuccinylase • Dihydrolipoyl dehydrogenase (identical to PDC) • Five coenzymes used - TPP, CoA-SH, Lipoic acid, NAD+, FAD

  30. Like pyruvate dehydrogenase, -ketoglutarate dehydrogenase is a multienzyme complex – consisting of -ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase. The complex uses five different coenzymes.

  31. 19.4 – How Is Oxaloacetate Regenerated to Complete the TCA Cycle? Succinyl-CoA SynthetaseA substrate-level phosphorylation

  32. Thioester [Succinyl-P] [Phospho-histidine] GTP • A nucleoside triphosphate is made GTP + ADP  ATP + GDP (nucleotide diphosphate kinase) • Its synthesis is driven by hydrolysis of a CoA ester • The mechanism involves a phosphohistidine Figure 19.11 The mechanism of the succinyl-CoA synthetase reaction.

  33. Completion of the TCA Cycle – Oxidation of Succinate to Oxaloacetate • This process involves a series of three reactions • These reactions include: • Oxidation of a single bond to a double bond (FAD/FADH2) • Hydration reaction • Oxidation of the resulting alcohol to a ketone (NAD+/NADH) • These reactions will be seen again in: • Fatty acid breakdown and synthesis • Amino acid breakdown and synthesis

  34. Succinate Dehydrogenase The oxidation of succinate to fumarate (trans-)

  35. Succinate Dehydrogenase • A membrane-bound enzyme that is actually part of the electron transport chain 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 pathway (chapter 20) • FAD is covalently bound to the enzyme Figure 19.12 The covalent bond between FAD and succinate dehydrogenase links the C-8a carbon of FAD and the N-3 of a His residue of the enzyme.

  36. Succinate Dehydrogenase • Succinate oxidation involves removal of H atoms across a C-C bond, rather than a C-O or C-N bond • The reaction is not sufficiently exergonic to reduce NAD+ • Contains iron-sulfur cluster Succinate Dehydrogenase contains three types of Fe-S centers – a 4Fe-4S center, a 3Fe-4S center, and a 2Fe-2S center.

  37. Fumarase Hydration across the double bond • Catalyzes the trans-hydration of fumarate to form L-malate • trans-addition of the elements of water across the double bond

  38. Possible mechanisms are shown in Figure 19.13

  39. Malate Dehydrogenase An NAD+-dependent oxidation • Completes the Cycle by Oxidizing Malate to Oxaloacetate • The carbon that gets oxidized is the one that received the-OH in the previous reaction • This reaction is very endergonic, with a Go' of +30 kJ/mol • The concentration of oxaloacetate in the mitochondrial matrix is quite low

  40. Steric Preferences in NAD+ Dependent Dehydrogenases

  41. 19.5 – What Are the Energetic Consequences of the TCA Cycle? One acetate through the cycle produces two CO2, one ATP, four reduced coenzymes Acetyl-CoA + 3 NAD+ + FAD + ADP + Pi + 2 H2O → 2 CO2 + 3 NADH + 3 H+ + FADH2 + ATP +CoASH DG0’ = -40kJ/mol Glucose + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi + 2 H2O → 6 CO2 + 10 NADH + 10 H+ + 2 FADH2 + 4 ATP NADH + H+ + 1/2 O2 + 3 ADP + 3 Pi → NAD+ + 3ATP + H2O FADH2 + 1/2 O2 + 2 ADP + 2 Pi → FAD + 2ATP + H2O

  42. The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle • Neither of the carbon atoms of a labeled acetate unit is lost as CO2 in the first turn of the cycle • Carbonyl C of acetyl-CoA turns to CO2 only in the second turn of the cycle (following entry of acetyl-CoA ) • Methyl C of acetyl-CoA survives two cycles completely, but half of what's left exits the cycle on each turn after that.

  43. The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle Figure 19.15The fate of the carbon atoms of acetate in successive TCA cycles. (a) The carbonyl carbon of acetyl-CoA is fully retained through one turn of the cycle but is lost completely in a second turn of the cycle.

  44. The Carbon Atoms of Oxaloaceate in the TCA Cycle • Both of the carbonyl carbons of oxaloaceate are lost as CO2, but the methylene and carbonyl carbons survive through the second turn • The methylene carbon survives two full turns of cycle • The carbonyl carbon is the same as the methyl carbon of acetyl-CoA

  45. 19.6 – Can the TCA Cycle Provide Intermediates for Biosynthesis? The products in TCA cycle also fuel a variety of biosynthetic processes • α-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

  46. Figure 19.16The TCA cycle provides intermediates for numerous biosynthetic processes in the cell.

  47. Citrate can be exported from the mitochondria and then broken down by citric lyase to yield acetyl-CoA and oxaloacetate (chapter 24) • Oxaloacetate is rapidly reduced to malate • Malate can be transported into mitochondria or oxidatively decarboxylated to pyruvate by malic enzyme • Oxaloacetate can also be decarboxylated to yield PEP

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