Harvesting Electrons from the Citric Acid Cycle CHAPTER 19 Harvesting Electrons from the Citric Acid Cycle

Harvesting electrons from the citric acid cycle Enzymes of the cycle are in the mitochondria of eukaryotes Energy of the oxidation reactions is largely conserved as reducing power (stored electrons) Coenzymes reduced: NAD+ NADH FAD FADH2 The net effect of the cyclic pathway is the complete oxidation of an acetyl group to 2 CO2 and the transfer of electrons to the above coenzymes.

Figure 19.1: An overview of the citric acid cycle 8 electrons are stored 2 carbon units are converted to CO2 and 1 ATP is made in the cycle

Figure 19.2: Harvested electrons will be used to drive a Proton Pump

1. Citrate Synthase Citrate is formed from the acetyl CoA and oxaloacetate Methyl carbon from acetyl group reacts with carbonyl of oxaloacetate - This is the only cycle reaction with C-C bond formation

1. Citrate Synthase The induced fit model revisited: Side reaction could be the hydrolysis of acetyl CoA A homodimeric protein Oxaloacetate caused a major rearrangement that creates a binding site for acetyl CoA reduces the possibility of side reactions

2. Aconitase Aconitase catalyzes a near equilibrium reaction Citrate is not a chiral molecule Elimination of H2O from citrate to form C=C bond of cis-aconitate A stereospecific addition of H2O to cis-aconitate forms 2R,3S-isocitrate

3. Isocitrate Dehydrogenase Oxidative decarboxylation of isocitrate to a-ketoglutarate (a metabolically irreversible reaction) One of four oxidation-reduction reactions of the cycle Hydride ion from the C-2 of isocitrate is transferred to NAD+ to form NADH. (unstable)

4. The a-Ketoglutarate Dehydrogenase Complex Complex is very large and is similar to the pyruvate dehydrogenase complex - E1 – a-ketoglutarate dehydrogenase (with TPP) - E2 – succinyltransferase (with a flexible lipoamide prosthetic group) - E3 – dihydrolipoamide dehydrogenase (with FAD) Thiamine 2nd reducing equivalence

Succinyl-CoA Synthetase The beginning of regenerating oxaloacetate The high free energy stored in the thioester bond of succinyl CoA is conserved as GTP (ATP equivalence): Figure 13.8 Proposed mechanism of succinyl–CoA synthetase. Phosphate displaces CoA from a bound succinyl CoA molecule, forming the mixed acid anhydride succinyl phosphate as an intermediate. The phosphoryl group is then transferred from succinyl phosphate to a histidine residue of the enzyme to form a relatively stable covalent phosphoenzyme intermediate. Succinate is released, and the phosphoenzyme intermediate transfers its phosphoryl group to GDP (or ADP, depending on the organism), forming the nucleoside triphosphate product.

Succinyl-CoA Synthetase The beginning of regenerating oxaloacetate The high free energy stored in the thioester bond of succinyl CoA is conserved as GTP (or ATP) The reaction takes place in four steps: Figure 19.5 High energy thioester bond Figure 13.8 Proposed mechanism of succinyl–CoA synthetase. Phosphate displaces CoA from a bound succinyl CoA molecule, forming the mixed acid anhydride succinyl phosphate as an intermediate. The phosphoryl group is then transferred from succinyl phosphate to a histidine residue of the enzyme to form a relatively stable covalent phosphoenzyme intermediate. Succinate is released, and the phosphoenzyme intermediate transfers its phosphoryl group to GDP (or ADP, depending on the organism), forming the nucleoside triphosphate product.

6. The Succinate Dehydrogenase (SDH) Complex The active site is made of two different subunits One subunit contains 4 Fe-S cluster The other contains covalently bound FAD The SDH dimer is attached to the inner mitochondrial membrane via two membrane polypeptides - This is in contrast to other enzymes of the CA cycle which are dissolved in the mitochondrial matrix The transfer of two electrons occurs from succinate  FADH2  ubiquinone (Q), a lipid soluble mobile carrier of electrons - The reduced ubiquinone (QH2) is released as a mobile product Succinate-Q reductase in the OP steps Q QH2 + FAD

7. Fumarase A near equilibrium trans addition of water to the double bond of fumarate

8. Malate Dehydrogenase A near equilibrium inter-conversion that is dependent on NAD+ Oxidation of malate to oxaloacetate, with transfer of electrons to NAD+ to form NADH.

Figure 19.6 The two carbons that enter the cycle are NOT the same to leave as CO2 FAD + QH2 Q

The overall net reaction can be simplified as follows: The OH- is ultimately donated by a phosphate group From the 8 electrons and 7 H+: 6 electrons are transferred to 3 NAD+ along with 3 H+ 2 electrons are transferred to Q along with 2 H+ 2 H+ are released per cycle. The two CO2 molecules do NOT come directly from the acetyl group added to CoA

Reduced Coenzymes Fuel the Production of ATP Each acetyl CoA entering the cycle nets: 1. 3 NADH 2. 1 QH2 3. 1 GTP (or 1 ATP) Oxidation of each NADH yields 2.5 ATP Oxidation of each QH2 yields 1.5 ATP Complete oxidation of 1 acetyl CoA = 10 ATP Oxidative phosphorylation

Glucose degradation via glycolysis, citric acid cycle and oxidative phosphorylation Figure 13.10 ATP production from the catabolism of one molecule of glucose by glycolysis, the citric acid cycle, and reoxidation of NADH and QH2 The complete oxidation of glucose leads to the formation of up to 32 molecules of ATP.

Regulation of the citric acid cycle Figure 19.7 Regulation of the citric acid cycle occurs mainly in two reactions: 1. isocitrate dehydrogenase (isocitrate  a-ketoglutarate) 2. a-ketogluterate dehydrogenase complex (a-ketoglutarate  succinyl CoA) ADP signifies the need for more energy Slowing isocitrate dehydrogenase allows for buildup of citrate which also Slows PFK and citrate can be also used for biosynthesis.

Figure 19.8 Routes leading to and from the CA cycle

Figure 19.9: Pyruvate carboxylase supplies oxaloacetate for the citric acid cycle When energy demands are high oxaloacetate is diverted to gluconeogenesis When energy demands are low oxaloacetate is shuttled into citric acid cycle

The Glyoxylate Cycle Pathway for the formation of glucose from noncarbohydrate precursors in plants, bacteria and yeast (few animals) Glyoxylate cycle leads from 2-carbon compounds to glucose -- Glyoxylate (a 2 C compound) combines with the acetyl group in acetyl CoA to generate malate leads to glucose formation In most animals, acetyl CoA is NOT a carbon source for the net formation of glucose (2 carbons of acetyl CoA enter CA cycle, 2 carbons are released as 2 CO2 Glyoxylate cycle provides an anabolic alternative for acetyl CoA metabolism (Allows for the formation of glucose from acetyl CoA) This cycle is active in oily seed plants (Stored seed oils are converted to carbohydrates during germination) Germination is the process in which a plant or fungus emerges from a seed or spore, respectively, and begins growth

The glyoxylate cycle bypasses the two decarboxylation steps Figure 19.10 The glyoxylate cycle bypasses the two decarboxylation steps of the CA cycle, conserving the two carbon atoms as glyoxylate for synthesis of glucose Germinating seeds use this Pathway to synthesize sugar (glucose) from oil (triacylglycerols) Figure 13.14 Glyoxylate pathway. Isocitrate lyase and malate synthase are the two enzymes of the pathway. When the pathway is functioning, the acetyl carbon atoms of acetyl CoA are converted to malate rather than oxidized to CO2. Malate can be converted to oxaloacetate which is a precursor in gluconeogenesis. The succinate produced in the cleavage of isocitrate is oxidized to oxaloacetate to replace the four-carbon compound consumed in glucose synthesis.

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