Journal
BIOCHEMISTRY
Volume 48, Issue 33, Pages 7878-7891Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi9006447
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Funding
- National Institutes of Health [GM 33851]
- National Science Foundation [MCB 0936108]
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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 (lie processes that occur in the condensation half-reaction performed by Thermoplasma acidophilum citrate synthase (7)CS) 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.
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