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 [(18)O(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 [(18)O(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 (18)O uptake from H(2)(18)O 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.
Selmer
T
T
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, D-35032 Marburg, Germany. selmer@mailer.uni-marburg.de
Buckel
W
W
D001421:Bacteria, Anaerobic / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D015256:Coenzyme A-Transferases / Q000378:metabolism*; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000378:metabolism; D006868:Hydrolysis; D013058:Mass Spectrometry; D010100:Oxygen / Q000378:metabolism*
D001426:Bacterial Proteins; D018698:Glutamic Acid; D015256:Coenzyme A-Transferases; C032430:glutaconate CoA-transferase; D010100:Oxygen