The partial substrate dethiaacetyl-coenzyme A mimics all critical carbon acid reactions in the condensation half-reaction catalyzed by Thermoplasma acidophilum citrate synthase.
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.
Kurz
Linda C
LC
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Constantine
Charles Z
CZ
Jiang
Hong
H
Kappock
T Joseph
TJ
D000105:Acetyl Coenzyme A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004952:Esters; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007659:Ketones / Q000037:antagonists & inhibitors; D007700:Kinetics; D018716:Molecular Mimicry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013552:Swine; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics; D014364:Tryptophan / Q000235:genetics
D004952:Esters; D007659:Ketones; C105313:dethiaacetyl coenzyme A; D001224:Aspartic Acid; D000105:Acetyl Coenzyme A; D014364:Tryptophan; D002950:Citrate (si)-Synthase