CITRIC - The Citric Acid Cycle Bryant Miles I. Citrate...

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The Citric Acid Cycle Bryant Miles I. Citrate Synthase The first reaction of the citric acid cycle is the condensation of acetyl-CoA and oxaloacetate to form citrate and CoA-SH. The enzyme that catalyzes this reaction is called citrate synthase. Δ G o ’ = -32.2 kJ/mol The change in free energy based on the steady state concentrations of oxaloacetate, acetyl-CoA and citrate in the mitochondria of isolated from pig hearts is: Δ G = -53.9 kJ/mol A very exergonic reaction and irreversible. The mechanism of citrate synthase is shown to the left. In the active site of the enzyme we have two histidines and an aspartate which function as general acids and bases during catalysis. The first step is to generate an enol of acetyl-CoA. Asp-375 functions as a general base abstracting a proton for the methyl group of Acetyl-CoA. His-274 concertedly functions as a general acid donating a proton to the carbonyl to form the enol. The enol generated in the first step is converted into a nucleophile by the abstraction of the enol hydrogen by His- 274 now functioning as a general base. The electrons of the double bond attack the electrophilic center of the ketone of oxaloactete. His-320 functions in concert as a general acid donating its proton to the carbonyl oxygen of oxaloacetate to form citryl-CoA. Citryl-CoA spontaneously hydrolyses while it is still bound to the active site to generate coenzyme A and citrate. H 3 CC O SCoA CO 2 - CO CH 2 CO 2 - CH 2 CC O 2 - CH 2 CO 2 - CO 2 - HO + CoASH + H + CO 2 - O H 2 C - O 2 C His 274 CH 2 N N H H + C C O CoAS H H H O - O Asp 375 CO 2 - O H 2 C - O 2 C His 274 CH 2 N N H H C C O CoAS H H H O O Asp 375 His 320 CH 2 N N H H + CO 2 - OH H 2 C - O 2 C CH 2 C O CoAS His 320 CH 2 N N H H O O Asp 375 His 274 CH 2 N N H H H 2 O CH 2 CC O 2 - CH 2 CO 2 - CO 2 - HO
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Citrate synthase is a homodimer with symmetry as you can see to the left. It has a sequential order kinetic mechanism. First the enzyme binds oxaloacetate which induces the large conformational change shown (b). This is yet another example of induced fit. The conformation change induced by oxaloacetate binding creates the acetyl- CoA binding site and seals oxaloacetate form the aqueous solvent. Now that you are no longer novices regarding metabolic strategies, you probably already have a sense that citrate synthase is going be allosterically regulated. It is the first step of a metabolic pathway. It catalyzes an irreversible step in the pathway. It is a homodimer with symmetry. And your intuitions are correct. This enzyme is a site for allosteric regulation. One of the products of the citric acid cycle is NADH which allosterically inhibits the enzyme. Succinyl- CoA, the product of the fifth step of the cycle also inhibits. II. Aconitase
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CITRIC - The Citric Acid Cycle Bryant Miles I. Citrate...

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