Citric acid cycle Ch. 16

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1. Citrate synthase Synthases are lyases working in reverse 1/17/2019 Remember a lyase is an enzyme that catalyzes the cleavage of substrates in simple elimination reactions that are NOT oxidation-reduction reactions and are NOT coupled to ATP hydrolysis. Synthases belong to the lyase category; you will see that synthetases belong to the ligase category. Learn the terms now! 1/17/2019 S.A. McFarland © 2007

Sample problem The standard free energy change for the citrate synthase reaction is -31.5 kJ/mol. Normally, you might expect such a large -ΔG to be coupled with the synthesis of ATP. However, no ATP (or GTP) is produced during this step. Give a possible explanation. Concentration of oxaloacetate is normally very low. 1/17/2019 S.A. McFarland © 2007

1. Citrate synthase Oxaloacetate binding causes 18o rotation of smaller domain, which closes the cleft Conformational change creates binding site for acetyl CoA Sequential binding very important in preventing wasteful hydrolysis of acetyl CoA Formation of citryl CoA thioester is followed by hydrolysis In gram+ bacteria and eukaryotes, citrate synthase is a dimeric protein composed of 2 identical subunits. Each subunit of the mammalian enzyme has 2 distinct domains; a small, flexible domain on the outer surface and a larger domain that forms the core of the protein. 1/17/2019 S.A. McFarland © 2007

A similar conformational change occurs for which enzyme in glycolysis? 1/17/2019 S.A. McFarland © 2007

1. Enol of acetyl-CoA is generated in rate-limiting step 2. Citryl-CoA is formed in a concerted acid-base catalyzed step (one of few enzymes that can form a C-C bond without a metal ion cofactor 3. Citryl-CoA is hydrolyzed to citrate and CoA 1/17/2019 S.A. McFarland © 2007

Acetyl CoA thioester is near the active site for the hydrolysis of citryl CoA thioester Concentration of oxaloacetate may be very low relative to concentration of acetyl CoA Considering the reaction mechanism above, provide a rationale behind the sequential binding of acetyl-CoA after oxaloacetate. 1/17/2019 S.A. McFarland © 2007

2. Aconitase Reversible isomerization of citrate and isocitrate Stereospecific addition of H2O to cis-aconitate to form 2R,3S-isocitrate 1/17/2019 S.A. McFarland © 2007

2. Aconitase [4Fe-4S] iron-sulfur cluster holds substrate in a preferred conformation Iron-sulfur clusters usually participate in redox processes; aconitase is an intriguing exception. 1/17/2019 S.A. McFarland © 2007

2. Aconitase [4Fe-4S] iron-sulfur cluster Addition of H2O across the double bond of cis-aconitate could potentially form four diastereomers; but only one is formed. 1/17/2019 S.A. McFarland © 2007

Sample problem The CAC is oxidative (as are all catabolic processes). Study the structure of citrate and propose a reason for the existence of aconitase. Tertiary hydroxyl cannot be oxidized. Make a beta-keto acid. 1/17/2019 S.A. McFarland © 2007

3. Isocitrate dehydrogenase Oxidative decarboxylation of isocitrate to α-ketoglutarate Oxidation of 2o alcohol to a ketone followed by decarboxylation of carboxyl group β to ketone First generation of CO2 and NADH in the CAC Note that this CO2 began the cycle as part of oxaloacetate (not of acetyl-CoA) Enzyme requires Mn2+ or Mg2+ as a cofactor 1/17/2019 S.A. McFarland © 2007

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Sample problem Why is it desirable to move the C3 hydroxyl group in citrate to C2? You have now formed a beta ketoacid which can decarboxlate to produce CO2. 1/17/2019 S.A. McFarland © 2007

Sample problem What is the role of Mn2+ in this particular reaction? State similar examples. It acts as a Lewis acid, polarizing the newly formed ketone. aldolase (Class II) and yeast alcohol dehydrogenase (we may not have emphasized the latter). 1/17/2019 S.A. McFarland © 2007

4. α-Ketoglutarate dehydrogenase complex Oxidative decarboxylation of an α-ketoacid 2nd CO2 and NADH produced in this reaction Mechanism is similar to PDH complex Product is a “high-energy” thioester 1/17/2019 S.A. McFarland © 2007

4. α-Ketoglutarate dehydrogenase complex Oxidative decarboxylation of an α-ketoacid Similar to pyruvate dehydrogenase complex Same coenzymes, identical mechanisms E1 - a-ketoglutarate dehydrogenase (with TPP) E2 - succinyltransferase (with flexible lipoamide prosthetic group) E3 - dihydrolipoamide dehydrogenase (with FAD) Exothermic (ΔGo’= -33.5 kJ/mol) 1/17/2019 S.A. McFarland © 2007

End of first phase 2 carbons have been introduced as acetyl-CoA Phase I: addition of carbons, loss of carbons Phase II: regeneration of oxaloacetate 2 carbons have been introduced as acetyl-CoA 2 carbons have been lost as CO2 2 carbons lost are not the same as 2 that are introduced remaining reactions convert 4C succinyl-CoA to 4C oxaloacetate 1/17/2019 S.A. McFarland © 2007

5. Succinyl-CoA synthetase Note the difference between a synthetase and synthase Named for reverse reaction & is called a synthetase b/c it combines 2 molecules by coupling to hydrolysis of a nucleoside triphosphate Free energy in thioester bond of succinyl CoA is conserved as GTP (or ATP in plants, some bacteria) Only example of substrate level phorphorylation in CAC Rxn 1 used a synthase and Rxn 5 uses a synthetase. 1/17/2019 S.A. McFarland © 2007

Reminiscent of passing a hot potato 1/17/2019 S.A. McFarland © 2007

Step 8 of glycolysis; phosphoglycerate mutase. In what other metabolic reaction have you seen the involvement of a phospho-His? 1/17/2019 S.A. McFarland © 2007

6. Succinate dehyrogenase complex (SDH) A stereospecific dehydrogenation Located on the inner mitochondrial membrane of eukaryotes (other components of CAC are dissolved in the matrix) Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters Uses FAD as an oxidant Dehydrogenation is stereospecific; only the trans isomer is formed Near equilibrium rxn Substrate analog malonate is a competitive inhibitor of the SDH complex 1/17/2019 S.A. McFarland © 2007

Sample problem Malonate is a structural analog of succinate and inhibits the SDH complex. What type of inhibition do you suspect? Predict the effects on Km and vmax and draw the corresponding Lineweaver-Burke plot. 1/17/2019 S.A. McFarland © 2007

6. Succinate dehyrogenase complex (SDH) SDH contains FAD as a covalently bound prosthetic group FAD functions to oxidize alkanes Reoxidation of FADH2 occurs when electrons are passed to the mitochondrial electron transport chain 1/17/2019 S.A. McFarland © 2007

7. Fumarase Another stereospecific hydration Hydration of double bond to form malate Reaction proceeds through a carbanion transition state Near equilibrium reaction 1/17/2019 S.A. McFarland © 2007

8. Malate dehyrogenase Regeneration of oxaloacetate Hydroxyl group of malate oxidized by NAD+ Hydride transfer occurs by same mechanism used in LDH and alcohol DH ΔGo’= +29.7 kJ/mol Concentration of oxaloacetate at equilibrium (and under cellular conditions) is very low (<1 mM) Remember that low concentrations of oxaloacetate necessitates a large -ΔGo’ for the first CAC reaction 1/17/2019 S.A. McFarland © 2007

CAC summary acetyl-CoA + 3 NAD+ + FAD + GDP + Pi CoA + 3 NADH + FADH2 + GTP + 2 CO2 1/17/2019 S.A. McFarland © 2007

Regulation of the CAC Availability of substrates Capacity to generate energy for cellular needs is closely regulated Availability of substrates Need for CAC intermediates as biosynthetic precursors Demand for ATP 1/17/2019 S.A. McFarland © 2007

Energy-generating capacity ~38 ATP per glucose under aerobic conditions (when CAC engaged) Substrate-level phosphorylation Glycolysis (2) CAC (2) Oxidative phosphorylation Glycolysis (6) PDH complex (6) CAC (22) Note that 38 is only an approximation; some textbooks list 32. Also note that one turn of the CAC produces 12 ATP (or 10 in other textbooks). 1/17/2019 S.A. McFarland © 2007

Energy-generating capacity ~2 ATP per glucose under anaerobic conditions (when CAC NOT engaged) Remember that NAD+ is regenerated by pyruvate reduction (homolactic fermentation) Note that 38 is only an approximation; some textbooks list 32. Also note that one turn of the CAC produces 12 ATP (or 10 in other textbooks). 1/17/2019 S.A. McFarland © 2007

Regulation of CAC Entry Rate-controlling enzymes PDH complex Citrate synthase Isocitrate DH α-Ketoglutarate DH complex 1/17/2019 S.A. McFarland © 2007

Regulation of PDH complex Decarboxylation of pyruvate is irreversible No other pathways in mammals for synthesis of acetyl-CoA from pyruvate Therefore, crucial that PDH reaction by precisely controlled Product inhibition Covalent modification 1/17/2019 S.A. McFarland © 2007

Precise control of PDH complex Product inhibition 1/17/2019 S.A. McFarland © 2007

Precise control of PDH complex Product inhibition Compete with NAD+ and CoA for binding sites on their respective enzymes Drive reversible transacetylase (E2) and dihydrolipoyl DH (E3) reactions backward High [NADH]/[NAD+] and [acetyl-CoA]/[CoA] ratios maintain E2 in the acetylated form Acetylated form incapable of accepting hydroxyethyl group from TPP, which ties up TPP in its hydroxyethyl form Decreased rate of pyruvate decarboxylation 1/17/2019 S.A. McFarland © 2007

Sample problem What renders the pyruvate DH (E1) reaction irreversible? 1/17/2019 S.A. McFarland © 2007

Precise control of PDH complex Covalent modification by phosphorylation/dephosphorylation of E1 In eukaryotes, NADH and acetyl-CoA also activate the pyruvate DH kinase Phosphorylation of a Ser inactivates the PDH complex Insulin, the hormone that signals fuel abundance, reverses inactivation by activating pyruvate DH phosphatase (removal of phosphate group) Insulin also stimulates glycogen synthesis; thus, insulin responds to elevated blood [glucose] by promoting synthesis of acetyl-CoA and glycogen There are other regulators of the PDH system, but we will not cover them. 1/17/2019 S.A. McFarland © 2007

Classify the pyruvate DH phosphatase reaction. 1/17/2019 S.A. McFarland © 2007

Regulation of rate-controlling enzymes Estimates are from heart muscle or liver tissue. 1/17/2019 S.A. McFarland © 2007

Regulation of rate-controlling enzymes Flux is controlled primarily by 3 simple mechanisms Substrate availability Product inhibition Competitive feedback inhibition by subsequent intermediates 1/17/2019 S.A. McFarland © 2007

Regulation of rate-controlling enzymes Flux is controlled primarily by 3 simple mechanisms No single flux-control point Substrate availability Product inhibition Competitive feedback inhibition by subsequent intermediates 1/17/2019 S.A. McFarland © 2007

Additional regulation Allosteric Isocitrate DH (ADP & Ca2+ activate, ATP inhibits) α-Ketoglutarate DH complex (Ca2+ activates) Covalent E. coli isocitrate DH Why might Ca2+ be an allosteric activator of some CAC enzymes? 1/17/2019 S.A. McFarland © 2007

Additional regulation Allosteric Isocitrate DH (ADP & Ca2+ activate, ATP inhibits) α-Ketoglutarate DH complex (Ca2+ activates) Covalent E. coli isocitrate DH Why might Ca2+ be an allosteric activator of some CAC enzymes? Ca2+, the signal that stimulates muscle contraction, also stimulates production of ATP to fuel it. 1/17/2019 S.A. McFarland © 2007

Allosteric regulation 1/17/2019 S.A. McFarland © 2007

Covalent regulation E. coli isocitrate dehydrogenase 1/17/2019 S.A. McFarland © 2007

Summary 1/17/2019 S.A. McFarland © 2007

Inhibition of CAC Compound 1080 = fluoroacetate Suicide substrate- a substrate that is not itself toxic. The body metabolizes the substrate into something that is toxic. Compound 1080 used by ranchers throughout the world to control coyotes, rabbits, and fox (<2mg/kg body weight fatal). Symptoms are intoxication-like effects, convulsions, and then death. an example of a suicide substrate… 1/17/2019 S.A. McFarland © 2007

Inhibition of CAC Compound 1080 = fluoroacetate Ingestion results in elevated levels of citrate in the heart, kidney, and brain. If ingestion of fluoroacetate results in the build-up of citrate, what enzyme is inhibited? Propose a structure for the inhibitor. What type of inhibition do you suspect? Why are animals killed yet plants are unaffected by fluoroacetate? Suicide substrate- a substrate that is not itself toxic. The body metabolizes the substrate into something that is toxic. Compound 1080 used by ranchers throughout the world to control coyotes, rabbits, and fox (<2mg/kg body weight fatal). Symptoms are intoxication-like effects, convulsions, and then death. 1/17/2019 S.A. McFarland © 2007

Common themes Where in the CAC do you see this common theme? oxidation-hydration-oxidation Where in the CAC do you see this common theme? 1/17/2019 S.A. McFarland © 2007

Common themes oxidation-hydration-oxidation 1/17/2019 Different enzymes to facilitate the same “type” of reaction sequence. In both examples, we use an oxidation-hydration-oxidation sequence to oxidize a saturated carbon to a carbonyl group. 1/17/2019 S.A. McFarland © 2007