Transcript Kreb`s Cycle

Kreb’s Cycle
Chapter 16
Glycolysis:
6C Glu  3C Pyruvate x2
• Glu + 2NAD+ + 2 ADP + 2 Pi 
2 pyr + 2 NADH + 2 H+ + 2 ATP + 2 H2O
 D G’o= -85 kJ/mole
• 2 NADH  e- transport  ATP synth
• In cytosol
3C Pyruvate Product
• 2 C’s added to Coenzyme A (CoA)
– Acetate group
– Activates CoA (thioester)
• 1 C as CO2
Pyruvate Dehydrogenase
Complex (PDC)
• Catalyzes acetylation CoA
– Oxidative decarboxylation (LEO + cleave carboxylate)
Pyruvate Dehydrogenase
Complex (PDC)
• In mitochondria
• Sev copies of 3 associated enz’s
– Pyruvate dehydrogenase (E1)
– Dihydrolipoyl transacetylase (E2)
– Dihydrolipoyl dehydrogenase (E3)
• Book: mammalian PDC 5X size ribosome
– Bovine: circular arrangement
• 5 coenzymes
– Thiamine, riboflavin, niacin, pantothenate
• Two regulatory prot’s assoc’d
– Kinase, phosphatase
PDC E1: Pyruvate Dehydrogenase
• 24 copies in complex (E. coli)
• Coenzyme: thiamine pyrophosphate (TPP)
– From Vitamin B1
(Chpt 14)
• Pyruvate binds 
ethanolic grp att’d
to TPP
• CO2 released
• Ox’n to
acetaldehyde (att’d
as hydroxyethyl)
• Acetaldehyde
transferred to E2 of
PDC
(Chpt 14)
PDC E2: Dihydrolipoyl
Transacetylase
• “Core” of complex
• 24 copies (E. coli); 60
copies (bovine)
• Coenzyme: lipollysyl
– Molecular “arm”
– In ox’d form – 5
membered ring w/
disulfide
• Ethanolic grp to lipollysyl
– Ox’d  acetaldehyde
• -S-S- red’d to –SH HS- w/ ox’n to acetaldehyde
– Forms thioester
• Site of attack by CoASH
– Trans-esterification
–  AcetylCoA + dithiol lipoyl
PDC E3: Dihydrolipoyl
Dehydrogenase
• 12 copies att’d to E2 (E. coli)
• Coenzyme: FAD
– REMEMBER: Flavin nucleotide coenz’s bound to
enz’s
• (Nicotinamide nucleotides cofactors freer to dissociate)
– Used to reoxidize lipollysyl
• FAD red’d  FADH2
– Lipollysyl ox’d back to ring w/ disulfide
• FADH2 regen’d by NAD+ entry
– FADH2 ox’d  original FAD
– NAD+ red’d  NADH
• Leaves complex
• Where might it go?
PDC Summary
• 3 Enz’s closely assoc’d
– Book: “substrate channeling”
• Acetyl grp physically transferred
• Regulatory
– Both allosteric + covalently modified regulation
– E1 has kinase, phosphatase enz’s assoc’d
• Kinase phosphorylates, inactivates
• Phosphatase dephosphorylates, activates
• Assoc’d kinase allosterically controlled
– ATP stimulates
– Act’d kinase inactivates PDC
– So  [ATP]  ?? PDC??
– Modulators
• Inhibitory: ATP, NADH, acetyl CoA, fatty acids
– Why??
• Stimulatory: ADP/AMP, NAD+, pyruvate, CoA
– Why??
Kreb’s Cycle
• = Citric Acid Cycle = Tricarboxylic Acid Cycle =
TCA Cycle
• 2 C’s from pyr (as acetyl on acetylCoA)
• 2 C’s leave as CO2 (not same 2 C’s that entered)
• 4 redox rxn’s
– 3 NAD+  3 NADH; 1 FAD  FADH2
• Where will these go?
• 1 “high energy” phosphate bond formed
– 1 GDP  1 GTP (some cells 1 ADP  1 ATP)
– REMEMBER the name of this phosph’n?
• Oxaloacetate regen’d
• REMEMBER: 2 turns for each glu
• Up to 38 ATP/glu (>1160 kJ/mole avail)
• 1 step uses complex sim to PDC
(2C)
(6C)
(4C)
(6C)
(4C)
(5C)
(4C)
(4C)
(4C)
Acetyl CoA + Oxaloacetate
 Citrate + CoASH
Citrate Synthetase
• Condensation rxn
• CoASH regen’d
• Through CH3 of acetyl
• Transient intermediate: citroyl CoA
– Energy rel’d from cleavage acetylCoA
• Why? What grps impt to exergonic rxn?
• Oxaloacetate binds first
–  Conform’l change
– Now site for acetylCoA
• Modulators
– Availability of substrates
– Inhib’n w/  [citrate]
• What type of inhib’n?
•  [citrate] also inhibits PFK-1
– Where is PFK-1?
– What type of inhib’n would this be?
– Inhib’n w/  [ATP]
• Relieved w/  [ADP]
• Why?
– Inhib’n w/  [succinyl CoA]
• Feedback inhib’n
Citrate  Isocitrate
Aconitase
• Isomerization
• Through reversible
add’n H2O
• Cis-aconitate
intermediate
• Iron-sulfur center
• Prod rapidly
consumed in next
step
Isocitrate  a Ketoglutarate
+ CO2
Isocitrate Dehydrogenase
• Ox’n rxn (oxidative decarboxylation)
• Mn+2 coordinates/stabilizes intermediate
• NAD+ or NADP+ depending on isozyme
• Regulation
– Inhib’n w/  [ATP]
– Inhib’n w/  ratio [NADH]/[NAD+]
• Why?
a Ketoglutarate 
SuccinylCoA + CO2
a Ketoglutarate
Dehydrogenase Complex
• Identical rxn to PDC
• Sim E1, E2, E3 enzymes
– E1 aa’s differ, bind a ketoglutarate
specifically
• Same coenz’s
• Regulation
– Inhib’n w/  [succinyl CoA]
– Inhib’n w/  ratio [NADH]/[NAD+]
SuccinylCoA 
Succinate + CoASH
SuccinylCoA Synthetase
• Add’n Pi 
high energy
acyl
phosphate
intermediate
in enz active
site
• CoASH
released
• Phosphate transferred
to enz active site His
• GDP enters active
site; phosph’d  GTP
• Substrate level
phosph’n
• Book: GTP formed
transfers PO4 to ADP
later
Succinate  Fumarate
Succinate Dehydrogenase
• Membr-bound
– Euk’s – inner mitoch membr
– Prok’s – plasma membr
– Impt also in e- transport
• Iron-sulfur centers + FAD
– FAD may be cov’ly bound
• Malonate is competitive
inhibitor
Fumarate  L-Malate
Fumarase
• Hydration
trans
across db
– Enz
stereospecific
L-Malate  Oxaloacetate
L-Malate Dehydrogenase
• Substrate limited rxn
• Large + D G
– Why does the rxn go?
Cycle
• Complete w/ regen’n oxaloacetate
• Regulation through
– [substrate], [product]
– Coenz’s
– Nucleotide phosphates
– Other nutrient pathways
Catabolism/Anabolism Balanced
through Kreb’s Cycle
• Amphibolic
– Impt to both catabolism (breakdown) and
anabolism (build-up) of cell’s mol’s
– Catabolism of carbohydrates, FA’s, aa’s
through pyruvate, acetylCoA Kreb’s  ATP
– Anabolism by cycle intermediates  aa’s, fa’s,
lipids, purines/pyrimidines
• Balance of amphibolic pathways through
anapleurotic rxns
– Replenish cycle intermediates so TCA constant
– 4 impt rxns
– Synth oxaloacetate or malate from pyruvate or
phosphoenolpyruvate
• Where did you see these reactants?
– 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 suff to react w/ excess acetylCoA (from
excess pyr, from excess PEP)