1. Important updates No class on Monday, Nov. 19
Lab on Monday, Nov. 19 will pick up with the 1st week of GOT
Omitting the G1P lab completely
We’ll weight the lab you do better on more
Midterm 2 will be Friday, Nov. 30
Classes will go through Dec. 7 (which was the original date of our final exam)
No news regarding the new exam date yet (should know Nov. 15)
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2. Citric acid cycle
Ch. 16

3. 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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4. 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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5. Sample problem
What renders the pyruvate DH (E1) reaction irreversible?
Formation of CO2
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6. 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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7. Regulation of rate-controlling enzymes
Estimates are from heart muscle or liver tissue.
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8. 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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9. 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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10. 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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11. 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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12. Allosteric regulation
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13. Covalent regulation E. coli isocitrate dehydrogenase 4/22/2019
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14. Summary
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15. Inhibition of CAC Locoweed
Fluoroacetate (FA) is a natural product found in many South American, African, and Australian plants
Minor component in locoweed, found in the western US
Most mammals are fatally poisoned by less than 2 mg per kg bodyweight
Poisoned animals initially show intoxication-like behaviour followed by convulsions that result in death
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16. Inhibition of CAC Sodium fluoroacetate (Compound 1080)
Sodium fluoroacetate is sold as a commercial biocide (Compound 1080)
Used by ranchers to control coyotes, rabbits, and fox
Example of a “suicide substrate”, a substrate that is not itself toxic to cells but is metabolically converted to something toxic
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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17. Sample questions
Ingestion of fluoroacetate 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?
Fluorocitrate displays two physiological actions as a toxin: (1) it is a potent inhibitor of aconitase, and (2) it blocks citrate transport in and out of the mitochondria.
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19. 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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20. Common themes oxidation-hydration-oxidation 4/22/2019
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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21. CAC is amphibolic
Amphibolic indicates both catabolic and anabolic
At first glance pathways appear either catabolic (release and conservation of free energy) or anabolic (requirement for free energy)
CAC catabolic
Involves degradation
Major free-energy conservation system in most organisms
Intermediates required in only catalytic amounts to maintain degradative function of cycle
CAC anabolic
Several biosynthetic pathways use CAC intermediates as starting materials for anabolic reactions
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22. CAC intermediates Use and replenishment 4/22/2019
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23. Pathways that use Glucose biosynthesis (gluconeogenesis)
Fatty acid biosynthesis
Amino acid biosynthesis
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24. Amino acid biosynthesis
Reductive amination catalyzed by glutamate DH
Utilizes NADH or NADPH
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26. Pathways that replenish
Anaplerotic reactions
Anaplerotic rxns- literally, “filling-up” reactions
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27. Anaplerotic reactions
Replenishing CAC intermediates
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28. Pyruvate carboxylase Conversion of pyruvate to oxaloacetate
Most important of the anaplerotic reactions
Exists in mitochondria of animal cells but not in plants
Provides direct link between glycolysis and the CAC
Tetrameric protein of identical 1160-residue subunits
Each subunit has biotin prosthetic group covalently bound to lysine and a Mg2+ site
Absolute requirement for acetyl-CoA
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29. Sample problem
Acetyl-CoA is an activator of pyruvate carboxylase. When acetyl-CoA levels are high, pyruvate is converted to oxaloacetate. Why?
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30. Biotin Coenzyme that carries CO2
Imidazoline ring that is cis-fused to a tetrahydrothiophene ring bearing valerate side chain
Positions 1, 2, and 3 constitute a ureido group
Covalently attached to carboxylases by an amide linkage between valeryl carboxyl and ε-amino group of Lys
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31. Formation of biotinyl-enzyme
biotinyllysine = biocytin
Cleavage of ATP to form carboxyphosphate and ADP
Formation of a “high-energy” carboxyphosphate intermediate (activated from of CO2)
Exergonic reaction of biotin with CO2
Biotin-bound carbonyl is “activated” relative to bicarbonate (transfer does not involve further free-energy input)
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32. Conversion of pyruvate to oxaloacetate
biotinyllysine = biocytin
Activated carboxyl group is transferred from carboxybiotin to pyruvate in a 3-step reaction
Two reaction phases occur on different sites of same enzyme; the 14-Å arm of biocytin transfers the biotin ring between the two sites
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33. Movement of biocytin
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34. Sample problem
Biocytin (aka, biotinyllysine) functions similar to what other coenzyme? Where is this other coenzyme found and what is its function?
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35. All enter cells on same transporter
All become covalently attached to proteins by similar mechanisms
All provide flexible tether that allows bound reaction intermediates to move from one active site to another (substrate channeling)
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36. Sample problem
Biotin is a vitamin required by the human diet; it is abundant in many foods and is synthesized by intestinal bacteria. Biotin deficiency is rare, but can sometimes be caused by a diet rich in raw eggs. Considering that egg whites contain a large amount of the protein avidin, suggest a possible explanation.
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