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

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)

(6C)

(5C)

(4C)

(4C)

(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 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?

Ketoglutarate SuccinylCoA + CO2

Ketoglutarate Dehydrogenase Complex

• Identical rxn to PDC• Sim E1, E2, E3 enzymes

– E1 aa’s differ, bind 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

stereo-specific

L-Malate Oxaloacetate

L-Malate Dehydrogenase

• Substrate limited rxn• Large + 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)