Oxygen exchange between acetate and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans -: Implications for the mechanism of CoA-ester hydrolysis

The exchange of oxygen atoms between acetate, glutaryl-CoA, and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans was analyzed using [O-18(2)]acetate together with matrix-assisted laser desorption/ionization time of flight mass spectrometry of an appropriate undecapeptide, The exchange reaction was shown to be site-specific, reversible, and required both glutaryl-CoA and [O-18(2)]acetate. The observed exchange is in agreement with the formation of a mixed anhydride intermediate between the enzyme and acetate, In contrast, with a mutant enzyme, which was converted to a thiol ester hydrolyase by replacement of the catalytic glutamate residue by aspartate, no O-18 uptake from H-2 O-18 into the carboxylate was detectable. This result is in accord with a mechanism in which the carboxylate of aspartate acts as a general base in activating a water molecule for hydrolysis of the thiol ester intermediate, This mechanism is further supported by the finding of a significant hydrolyase activity of the wild-type enzyme using acetyl-CoA as substrate, whereas glutaryl-CoA is not hydrolyzed, The small acetate molecule in the substrate binding pocket may activate a water molecule for hydrolysis of the nearby enzyme-CoA thiol ester.