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The Citric Acid Cycle The Citric Acid Cycle • Three processes play central roles in aerobic metabolism •the citric acid cycle (this chapter) •electron transport (Chapter 17) •oxidative phosphorylation (Chapter 17) • Metabolism consists of •catabolism: catabolism: the oxidative breakdown of nutrients •anabolism: anabolism: the reductive synthesis of biomolecules • The citric acid cycle is amphibolic; amphibolic that is, it plays a role in both catabolism and anabolism The Citric Acid Cycle Pyruvate to Acetyl-CoA Pyruvate • Oxidation by NAD+ and formation of a thioester N AD + N AD H Acetyl -CoA Coenzyme A O CH3 CCOO - + CoA -SH + N AD + Pyruvate NAD H N AD + Citric acid cycle (8 steps) FAD H2 FAD GTP GDP N AD + N ADH CO 2 N AD N AD H CO 2 + Pyruvate dehydrogenase complex pyruvate dehydrogenase complex Coenzyme A O CH3 C-SCo A + CO 2 + N AD H Acetyl-CoA •this conversion requires NAD+, FAD, Mg2+, thiamine pyrophosphate, coenzyme A, and lipoic acid -1 •G° ’=-33. 4kJ• mol Pyruvate dehydrogenase complex Pyruvate dehydrogenase Dihydrolipoyl transacetylase Dihydrolipoyl dehydrogenase Pyruvate dehydrogenase kinase Pyruvate dehydrogenase phosphatase 1 Pyruvate to Acetyl-CoA • Step 1: pyruvate loses CO2 and HETPP is formed O CH 3 CCOO - + T PP Pyruvate pyruvate dehydrogenase OH CO 2 + CH 3 CH- TPP Hydroxyethyl-TPP Pyruvate to Acetyl-CoA • Step 2: •the hydroxyethyl group is oxidized and transferred to a sulfur atom of the reduced form of lipoamide •lipoamide is reduced to dihydrolipoamide O • Step 2 requires lipoic acid •the active form of lipoic acid is bound to the enzyme by an amide bond to the amino group of a lysine OH CH3 CH- TPP Hydroxyethyl-TPP C-N H- Enz + S S Lipoamide dihydrolipoyl transacylase COOH reduction oxidation S S Lipoic acid COOH HS SH Dihydrolipoic acid T PP Pyruvate to Acetyl-CoA • Step 3: the acetyl group is transferred to the O CH3 C S + • Step 4: oxidation of dihydrolipoamide O C-N H- Enz C-N H- Enz SH HS Dihydrolipoamide N AD + N AD H dihydrolipoyl transacylase O Acetyl-CoA Dihydrolipoamide O O SH CoA -SH + CH3 C-S Coenzyme A Dihydrolipoamide O C-N H- Enz + HS SH Pyruvate to Acetyl-CoA sulfhydryl group of coenzyme A O CoA -S-CCH3 O C-N H- Enz SH C-N H- Enz S S Dihydrolipoamide Where TCA cycle locate Lipoamide The Citric Acid Cycle • Step 1: condensation of acetyl-CoA with -1 oxaloacetate; G° ’=-32. 8kJ• mol O CH3 C-SCo A citrate Acetyl-CoA synthase + O C-COO CH2 -COO - CH2 -COO HO C-COO CH2 -COO - + CoA -SH Coenzyme A Citrate Oxaloacetate •citrate synthase is an allosteric enzyme, inhibited by NADH, ATP, and succinyl-CoA 2 The Citric Acid Cycle The Citric Acid Cycle • Step 2: dehydration and rehydration gives • Step 3: oxidation of isocitrate followed by isocitrate; catalyzed by aconitase CH2 -COO HO C-COO decarboxylation CH2 -COO - - C-COO - H C-COO CH- COO - CH2 -COO Citrate CH 2 -COO - N AD + CH 2 -COO - H C-COO HO CH- COO Isocitrate - HO CH- COO Isocitrate Aconitate •citrate is achiral; it has no stereocenter •isocitrate is chiral; it has 2 stereocenters and 4 stereoisomers are possible •only one of the 4 stereoisomers of isocitrate is formed in the cycle CH2 -COO - • isocitrate dehydrogenase is an allosteric enzyme; it is inhibited by ATP and NADH, activated by ADP and NAD+ The Citric Acid Cycle • Step 4: oxidative decarboxylation of - • Step 5: formation of succinate ketoglutarate to succinyl-CoA CH 2 -COO - CoA-SH CH2 O C-COO -Ketoglutarate N AD + CH2 -COO CH2 -ketoglutarate dehydrogenase complex + CO 2 O C SCoA Succinyl-CoA CH2 -COO - succinate dehydrogenase H FAD H 2 - OOC C C + GT P + CoA -SH Succinate •the two CH2-COO- groups of succinate are equivalent •this is the first energy-yielding step of the cycle •the overall reaction is slightly exergonic G 0' Succinyl-CoA + H2 O GDP + Pi Succinyl-CoA + GDP + Pi Succinate + CoA-SH GTP + H2 O (kJ ol -33.4 Succinate + CoA -SH + GTP -1 ) +30.1 -3.3 The Citric Acid Cycle • Step 6: oxidation of succinate to fumarate FAD CH 2 -COO CH 2 -COO - Succinyl-CoA The Citric Acid Cycle CH2 -COO - succinyl-CoA synthetase O C SCoA - •like pyruvate dehydrogenase, this enzyme is a multienzyme complex and requires coenzyme A, thiamine pyrophosphate, lipoic acid, FAD, and NAD+ -1 •G0’= -33. 4kJ• mol Succinate + GD P + Pi CH 2 N ADH CH2 -COO - CO 2 H C-H O C-COO -Ketoglutarate H C-COO O C-COO Oxalosuccinate The Citric Acid Cycle CH 2 -COO - N AD H isocitrate dehydrogenase • Step 7: hydration of fumarate COOH Fumarate H - C C COO- OOC H Fumarate H2 O HO CH- COO CH 2 -COO fumarase L-Malate 3 From Pyruvate to CO2 The Citric Acid Cycle Pyruvate dehydrogenase complex Pyruvate + CoA-SH + NAD + Acetyl-CoA + NADH + CO 2 + H + • Step 8: oxidation of malate N AD + N AD H HO CH- COO CH2 -COO L-Malate - malate dehydrogenase O C-COO Citric acid cycle Acetyl-CoA + 3NAD + + FAD + GDP + Pi + 2 H2 O 2 CO 2 + CoA-SH + 3NADH + 3H + + FAD H2 + GTP CH 2 -COO Oxaloacetate Pyruvate + 4NAD + + FAD + GDP + Pi + 2 H 2 O 3 CO 2 + 4NADH + FAD H2 + GTP + 4H + G? (kJol Pyruvate + CoA-SH + N AD + Acetyl-CoA + N AD H + CO 2 + H+ 1. Acetyl-CoA + Oxaloacetate + H2 O Citrate + CoA-SH + H+ Isocitrate 2. Citrate + 3. Isocitrate + N AD -Ketoglutarate + N AD H + CO 2 -33.4 4. -Ketoglutarate + N AD + + CoA-SH Succinyl-CoA + N AD H + CO 2 + H+ 5. Succinyl-CoA + GDP + Pi Succinate + GTP + CoA-SH 6. Succinate + FAD Fumarate + FAD H2 Malate 7. Fumarate + H2 O 8. Malate + N AD + Oxaloacetate + N AD H -33.4 -32.2 +6.3 -7.1 -3.3 ~0 -3.8 +29.2 -1 ) Control of the TCA Cycle • Three control points within the cycle •citrate synthase: inhibited by ATP, NADH, and succinyl CoA; also product inhibition by citrate •isocitrate dehydrogenase: dehydrogenase: activated by ADP and NAD+, inhibited by ATP and NADH •-ketoglutarate dehydrogenase complex: complex: inhibited by ATP, NADH, and succinyl CoA; activated by ADP and NAD+ • One control point outside the cycle •pyruvate dehydrogenase: inhibited by ATP and NADH; also product inhibition by acetyl-CoA Pyruvate + 4 N AD + + FAD + GDP + Pi -77.7 3 CO 2 + 4 N AD H + FAD H2 + GTP + 4 H+ Control of the CA Cycle Metabolic rate in different state • Cells in a resting metabolic state •need and use comparatively little energy •high ATP, low ADP imply high ATP/ADP ratio •high NADH, low NAD+ imply high NADH/NAD+ ratio • Cells in an active metabolic state •need and use more energy than resting cells •low ATP, high ADP imply low ATP/ADP ratio •low NADH, high NAD + imply low NADH/NAD+ ratio Figure 16.7 Conversion of pyruvate to acetyl-CoA 4 The Glyoxylate Cycle Glyoxylate Cycle CH 2 -COO - • Plants and some bacteria, but not animals, use a modification of the citric acid cycle to produce four-carbon dicarboxylic acids and eventually glucose •the glyoxylate cycle bypasses the two oxidative decarboxylations of the citric acid cycle •instead, it routes isocitrate via glyoxylate to malate •key enzymes in this cycle are isocitrate lyase and malate synthetase isocitrate lyase H C-COO HO CH- COO Isocitrate O O CH3 C-SCo A + HC- COO Acetyl-CoA CH 2 -COO CH 2 -COO Succinate malate synthetase Glyoxylate + O= C-COO H Glyoxylate OH CH- COO - + CoA -SH Coenzyme A CH 2 -COO Malate Glyoxylate Cycle The Glyoxylate Cycle • The glyoxylate cycle takes place • in plants: in glyoxysomes, specialized organelles devoted to this cycle • in yeast and algae: in the cytoplasm 5 The Glyoxylate Cycle TCA Cycle in Catabolism • The catabolism of proteins, carbohydrates, and • Helps plants grow in the dark •seeds are rich in lipids, which contain fatty acids •during germination, plants use the acetyl-CoA produced in fatty acid oxidation to produce oxaloacetate and other intermediates for carbohydrate synthesis •once plants begin photosynthesis and can fix CO 2, glyoxysomes disappear fatty acids all feed into the citric acid cycle at one or more points Carbohydrates Proteins Amino Acids Fatty Acids Pyruvate Acetyl-CoA -Ketoglutarate Succinyl-CoA Fumarate Malate Oxaloacetate CA Cycle in Catabolism intermediates of the citric acid cycle CA Cycle in Anabolism • The citric acid cycle is the source of starting materials for the biosynthesis of other compounds •examples: O - OOCCH 2 CH2 CCOO - transamination N H3 + - OOCCH 2 CH2 CH COO - -Ketoglutarate Glutamate O - OOCCH2 CCOO Oxaloacetate CA Cycle and Anabolism - transamination N H3 + - OOCCH2 CHCOO Aspartate The CA Cycle in Anabolism • If a component of the citric acid cycle is taken out for biosynthesis, it must be replaced •oxaloacetate, for example, is replaced by the carboxylation of pyruvate O CH3 CCOO - + CO 2 + A TP Pyruvate biotin pyruvate carboxylase O CH2 CCOO - + A DP + Pi COOOxaloacetate 6 The CA Cycle in Anabolism The CA Cycle in Anabolism The CA Cycle in Anabolism The CA Cycle in Anabolism • Lipid anabolism begins with acetyl-CoA and takes place in the cytosol •acetyl-CoA is produced mainly in mitochondria from catabolism of fatty acids and carbohydrates •an indirect transfer mechanism exists involving citrate Citrate + CoA-SH + ATP Acetyl-CoA + Oxaloacetate + ATP + Pi • the oxaloacetate thus formed provides a means for the production of the NADPH needed for biosynthesis Oxaloacetate + N AD H + H + Malate + N AD P+ Malate + N AD + Pyruvate + CO 2 + N AD PH + H + •the net effect of these two reactions is replacement of NADH by NADPH •while there is some NADPH produced by this means, its principal source is the pentose phosphate pathway • The anabolic reactions that produce amino acids and many other biomolecules begin with CA cycle molecules that are transported into the cytosol CA Cycle and Anabolism 7 細胞內所有代謝路徑 Nelson et al (2002) Principle of biochemistry (4e) p.560 8