(1) Citrate synthase catalyzes C-C bond formation between acetate and oxaloacetate Driven mainly by thioester hydrolysis.

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Presentation transcript:

(1) Citrate synthase catalyzes C-C bond formation between acetate and oxaloacetate Driven mainly by thioester hydrolysis

(1) Citrate synthase changes conformation in response to substrate binding (induced fit) Substrates bind sequentially: oxaloacetate, then acetyl-CoA

(1) Citrate synthase changes conformation in response to substrate binding (induced fit) chomp! Substrates bind sequentially: oxaloacetate, then acetyl-CoA

(1) In the rate-limiting step, acetyl-CoA is deprotonated to form an enolate

(1) The nucleophilic enolate attacks the carbonyl of oxaloacetate to yield citryl-CoA

(1) Hydrolysis of the “high-energy” thioester citryl-CoA makes the reaction irreversible A second conformational change allows hydrolysis at this step

(2) Aconitase catalyzes the stereospecific conversion of citrate to isocitrate

(2) Starting with a radio-labeled acetyl group yields label in only one position

(2) Aconitase can distinguish between the pro-R and pro-S substituents of citrate pro-S pro-R OH is always added here

(3) Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate ΔG'°= -21 kJ/mol

(3) In the first step, isocitrate is oxidized to oxalosuccinate, an unstable intermediate

(3) In the second step, oxalosuccinate is decarboxylated

(3) In the final step, the enolate rearranges and is protonated to form the keto product H+H+

(4) α-KG DH complex couples an oxidative decarboxylation with thioester formation ‘high-energy’ intermediate

(4) α-Ketoglutarate DH complex is similar to pyruvate DH complex EnzymePDH complexα-KGDH complexCofactors E1Pyruvate DHα-Ketoglutarate DHTPP E2Dihydrolipoyl transacetylase Dihydrolipoyl transsuccinylase Lipoic acid, Coenzyme A E3Dihydrolipoyl DH FAD, NAD How do E1, E2, and E3 differ between PDH complex and a-KG complex?

(5) Succinyl-CoA synthetase couples thioester hydrolysis with NTP synthesis ATP equivalent

(5) In the first step, the “high-energy” thio- ester becomes a “high-energy” anhydride ‘high-energy’ intermediate

(5) In the second step, the enzyme becomes the “high-energy” compound ‘high-energy’ intermediate

(5) In the third step, the phosphoryl group is transferred to form the “high-energy” NTP ATP equivalent

(6) The integral membrane protein succinate DH catalyzes succinate oxidation (tightly bound in enzyme)

(7) Fumarase catalyses the stereospecific conversion of fumarate to L-malate fumarase

(8) Malate dehydrogenase catalyzes the final step in regenerating oxaloacetate

The oxidation of acetyl-CoA to CO 2 in the TCA cycle generates energy currencies