1. Citric acid cycle
Ch. 16

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3. 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!
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4. Sample problem
The standard free energy change for the citrate synthase reaction is 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.
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5. 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.
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6. A similar conformational change occurs for which enzyme in glycolysis?
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7. 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
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8. 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.
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9. 2. Aconitase Reversible isomerization of citrate and isocitrate
Stereospecific addition of H2O to cis-aconitate to form 2R,3S-isocitrate
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10. 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.
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11. 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.
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12. 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.
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13. 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
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15. 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.
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16. 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).
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17. 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
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18. 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’= kJ/mol)
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19. 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
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20. 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.
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21. Reminiscent of passing a hot potato
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22. Step 8 of glycolysis; phosphoglycerate mutase.
In what other metabolic reaction have you seen the involvement of a phospho-His?
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23. 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
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24. 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.
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25. 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
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26. 7. Fumarase Another stereospecific hydration
Hydration of double bond to form malate
Reaction proceeds through a carbanion transition state
Near equilibrium reaction
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27. 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’= 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
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28. CAC summary acetyl-CoA + 3 NAD+ + FAD + GDP + Pi
CoA + 3 NADH + FADH2 + GTP + 2 CO2
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29. 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
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30. 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).
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31. 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).
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32. Regulation of CAC Entry Rate-controlling enzymes PDH complex
Citrate synthase
Isocitrate DH
α-Ketoglutarate DH complex
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33. 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
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34. Precise control of PDH complex
Product inhibition
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35. 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
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36. Sample problem
What renders the pyruvate DH (E1) reaction irreversible?
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37. 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.
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38. Classify the pyruvate DH phosphatase reaction.
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39. Regulation of rate-controlling enzymes
Estimates are from heart muscle or liver tissue.
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40. Regulation of rate-controlling enzymes
Flux is controlled primarily by 3 simple mechanisms
Substrate availability
Product inhibition
Competitive feedback inhibition by subsequent intermediates
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41. 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
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42. 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?
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43. 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.
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44. Allosteric regulation
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45. Covalent regulation E. coli isocitrate dehydrogenase 1/17/2019
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46. Summary
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47. 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…
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48. 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.
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49. 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?
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50. 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.
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