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The partial substrate dethiaacetyl-coenzyme A mimics all critical carbon acid reactions in the condensation half-reaction catalyzed by Thermoplasma acidophilum citrate synthase.


ABSTRACT: Citrate synthase (CS) performs two half-reactions: the mechanistically intriguing condensation of acetyl-CoA with oxaloacetate (OAA) to form citryl-CoA and the subsequent, slower hydrolysis of citryl-CoA that generally dominates steady-state kinetics. The condensation reaction requires the abstraction of a proton from the methyl carbon of acetyl-CoA to generate a reactive enolate intermediate. The carbanion of that intermediate then attacks the OAA carbonyl to furnish citryl-CoA, the initial product. Using stopped-flow and steady-state fluorescence methods, kinetic substrate isotope effects, and mutagenesis of active site residues, we show that all of the processes that occur in the condensation half-reaction performed by Thermoplasma acidophilum citrate synthase (TpCS) with the natural thioester substrate, acetyl-CoA, also occur with the ketone inhibitor dethiaacetyl-CoA. Free energy profiles demonstrate that the nonhydrolyzable product of the condensation reaction, dethiacitryl-CoA, forms a particularly stable complex with TpCS but not pig heart CS.

SUBMITTER: Kurz LC 

PROVIDER: S-EPMC2765034 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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The partial substrate dethiaacetyl-coenzyme A mimics all critical carbon acid reactions in the condensation half-reaction catalyzed by Thermoplasma acidophilum citrate synthase.

Kurz Linda C LC   Constantine Charles Z CZ   Jiang Hong H   Kappock T Joseph TJ  

Biochemistry 20090801 33


Citrate synthase (CS) performs two half-reactions: the mechanistically intriguing condensation of acetyl-CoA with oxaloacetate (OAA) to form citryl-CoA and the subsequent, slower hydrolysis of citryl-CoA that generally dominates steady-state kinetics. The condensation reaction requires the abstraction of a proton from the methyl carbon of acetyl-CoA to generate a reactive enolate intermediate. The carbanion of that intermediate then attacks the OAA carbonyl to furnish citryl-CoA, the initial pro  ...[more]

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