Catalytic mechanism of a C-C hydrolase enzyme: Evidence for a gem-diol intermediate, not an acyl enzyme

2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Es cherichia coli catalyses the hydrolytic cleavage of the extradiol ring fiss ion product on the phenylpropionate catabolic pathway and is a member of th e alpha/beta hydrolase family. The catalytic mechanism of this enzyme has p reviously been shown to proceed via initial ketonization of the dienol subs trate (Henderson, I. M. J., and Bugg,T. D. H. (1997) Biochemistry 36, 12252 -12258), followed by stereospecific fragmentation. Despite the implication of an active site serine residue in the alpha/beta hydrolase family, attemp ts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a C-14-labeled substrate yielded a stoichiometry of <1% coval ent intermediate, which could be accounted for by nonenzymatic processes. I n contrast, incorporation of 5-6% of two atoms of O-18 from (H2O)-O-18 into succinic acid was observed using the natural substrate, consistent with th e reversible formation of a gem-diol intermediate. Furthermore, time-depend ent incorporation of O-18 from (H2O)-O-18 into the carbonyl group of a nonh ydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presenc e of MhpC, consistent with enzyme-catalyzed attack of water at the ketone c arbonyl. These results favor a catalytic mechanism involving base-catalyzed attack of water, rather than nucleophilic attack of an active site serine. The implication of this work is that the putative active site serine in th is enzyme may have an alternative function, for example, as a base.