Kreb

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Krebs Cycle

• Chapter 16

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Glycolysis 6C Glu ? 3C
Pyruvate x2

• Glu 2NAD 2 ADP 2 Pi ?
  2 pyr 2 NADH 2 H
  2 ATP 2 H2O
• D Go -85 kJ/mole
• 2 NADH ? e- transport ? ATP synth
• In cytosol

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3C Pyruvate Product

• 2 Cs added to Coenzyme A (CoA)
• As acetate group
• Activates CoA (thioester)
• 1 C as CO2

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Pyruvate Dehydrogenase Complex (PDC)

• Catalyzes acetylation CoA
• Oxidative decarboxylation (LEO cleave
  carboxylate)

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Pyruvate Dehydrogenase Complex
(PDC)

• In mitochondria
• Sev copies of 3 associated enzs
• Pyruvate dehydrogenase (E1)
• Dihydrolipoyl transacetylase (E2)
• Dihydrolipoyl dehydrogenase (E3)

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• Book mammalian PDC 5X size ribosome
• Bovine circular arrangement
• 5 cofactors
• Thiamine, riboflavin, niacin, pantothenate
• Two regulatory proteins assocd
• Kinase, phosphatase

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PDC E1 Pyruvate Dehydrogenase

• 24 copies in complex (E. coli)
• Cofactor thiamine pyrophosphate (TPP)
• From Vitamin B1

(Chpt 14)
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• Pyr binds ? ethanolic grp attd to TPP
• CO2 released
• Oxn to acetaldehyde attd as hydroxyethyl
• Acetaldehyde transferred to E2 of PDC

(Chpt 14)
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PDC E2 Dihydrolipoyl Transacetylase

• Core of complex
• 24 copies (E. coli) 60 copies (bovine)
• Cofactor lipollysyl
• Molecular arm
• In oxd form 5 membered ring w/ disulfide

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• Ethanolic grp to lipollysyl
• Oxd ? acetaldehyde
• -S-S- redd to SH HS- w/ oxn to acetaldehyde
• Forms thioester
• Site of attack by CoASH
• Transesterification
• ? AcetylCoA dithiol lipoyl

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PDC E3 Dihydrolipoyl Dehydrogenase

• 12 copies attd to E2 (E. coli)
• Cofactor FAD
• REMEMBER Flavin nucleotide cofactors bound to
  enzs
• (Nicotinamide nucleotides cofactors freer to
  dissociate)
• Used to reoxidize lipollysyl

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• FAD redd ? FADH2
• Lipollysyl oxd back to ring w/ disulfide
• FADH2 regend by NAD entry
• FADH2 oxd ? original FAD
• NAD redd ? NADH
• Leaves complex
• Where might it go?

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PDC Summary

• 3 Enzs closely assocd
• Book substrate channeling
• Acetyl grp physically transferred
• Regulatory
• Both allosteric covalently modified regulation
• E1 has kinase, phosphatase enzs assocd
• Kinase phosphorylates, inactivates
• Phosphatase dephosphorylates, activates

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• Assocd kinase allosterically controlled
• ATP stimulates
• Actd kinase inactivates PDC
• So ? ATP ? ?? PDC??
• Modulators
• Inhibitory ATP, NADH, acetyl CoA, fatty acids
• Why??
• Stimulatory ADP/AMP, NAD, pyruvate, CoA
• Why??

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Krebs Cycle

• Citric Acid Cycle Tricarboxylic Acid Cycle
  TCA Cycle
• 2 Cs from pyr (as acetyl on acetylCoA)
• 2 Cs leave as CO2 (not same 2 Cs that entered)
• 4 redox rxns
• 3 NAD ? 3 NADH 1 FAD ? FADH2
• Where will these go?

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• 1 high energy phosphate bond formed
• 1 GDP ? 1 GTP (some cells 1 ADP ? 1 ATP)
• REMEMBER the name of this phosphn?
• Oxaloacetate regend
• REMEMBER 2 turns for each glu
• Up to 38 ATP/glu (gt1160 kJ/mole avail)
• 1 step uses complex sim to PDC

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Acetyl CoA Oxaloacetate ? Citrate CoASH
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Citrate Synthetase

• Condensation rxn
• CoASH regend
• Through CH3 of acetyl
• Transient intermediate citroyl CoA
• Energy reld from cleavage acetylCoA
• Why? What grps impt to exergonic rxn

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• Oxaloacetate binds first
• ? Conforml change
• Now site for acetylCoA

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• Modulators
• Availability of substrates
• Inhibn w/ ? citrate
• What type of inhibn?
• ? citrate also inhibits PFK-1
• Where is PFK-1?
• What type of inhibn would this be?
• Inhibn w/ ? ATP
• Relieved w/ ? ADP
• Why?
• Inhibn w/ ? succinyl CoA
• Feedback inhibn

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Citrate ? Isocitrate
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Aconitase

• Isomerization
• Through reversible addn H2O
• Cis-aconitate intermediate
• Iron-sulfur center
• Prod rapidly consumed in next step

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Isocitrate ? a Ketoglutarate CO2
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Isocitrate Dehydrogenase

• Oxn rxn (oxidative decarboxylation)
• Mn2 coordinates/stabilizes intermediate
• NAD or NADP depending on isozyme
• Regulation
• Inhibn w/ ? ATP
• Inhibn w/ ? ratio NADH/NAD
• Why?

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a Ketoglutarate ? SuccinylCoA CO2
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a Ketoglutarate Dehydrogenase Complex

• Identical rxn to PDC
• Similar E1, E2, E3 enzymes
• E1 aas differ, bind a ketoglutarate specifically
• Same cofactors
• Regulation
• Inhibn w/ ? succinyl CoA
• Inhibn w/ ? ratio NADH/NAD

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SuccinylCoA ? Succinate CoASH
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SuccinylCoA Synthetase

• Addn Pi ? high energy acyl phosphate
  intermediate in enz active site
• CoASH released

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• Phosphate transferred to enz active site His
• GDP enters active site phosphd ? GTP
• Substrate level phosphn results
• Book GTP formed transfers PO4 to ADP later

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Succinate ? Fumarate
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Succinate Dehydrogenase

• Membr-bound
• Euks inner mitoch membr
• Proks plasma membr
• Impt also in e- transport
• Iron-sulfur centers FAD
• FAD may be covly bound
• Malonate is competitive inhibitor

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Fumarate ? L-Malate
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Fumarase

• Hydration trans across db
• Enz stereo-specific

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L-Malate ? Oxaloacetate
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L-Malate Dehydrogenase

• Substrate limited rxn
• Large D G
• Why does the rxn go?

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Cycle

• Complete w/ regenn oxaloacetate
• Regulation through
• substrate, product
• Coenzymes
• Nucleotide phosphates
• Other nutrient pathways

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Catabolism/Anabolism Balanced through Krebs Cycle

• Amphibolic
• Impt to both catabolism (breakdown) and anabolism
  (build-up) of cells molecules
• Catabolism of carbohydrates, FAs, aas through
  pyruvate, acetylCoA? Krebs ? ATP
• Anabolism by cycle intermediates ? aas, fas,
  lipids, purines/pyrimidines

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• Balance of amphibolic pathways through
  anapleurotic rxns
• Replenish cycle intermediates so TCA remains
  constant
• 4 impt rxns
• Synth oxaloacetate or malate from pyruvate or
  phosphoenolpyruvate
• Where did you see these reactants?

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• If ??? glycolysis (so ??? PEP/pyr products), but
  not enough oxaloacetate to fuel cycle
• Cell can use excess PEP/pyr to make more
  oxaloacetate
• Now have sufficient to react w/ excess acetylCoA
  (from excess pyr, from excess PEP)

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