Catalytic Mechanism of a C−C Hydrolase Enzyme: Evidence for aGem-Diol Intermediate, Not an Acyl Enzyme

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Abstract
2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the hydrolytic cleavage of the extradiol ring fission product on the phenylpropionate catabolic pathway and is a member of the α/β hydrolase family. The catalytic mechanism of this enzyme has previously been shown to proceed via initial ketonization of the dienol substrate (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 α/β hydrolase family, attempts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a 14C-labeled substrate yielded a stoichiometry of 18O from H218O into succinic acid was observed using the natural substrate, consistent with the reversible formation of a gem-diol intermediate. Furthermore, time-dependent incorporation of 18O from H218O into the carbonyl group of a nonhydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presence of MhpC, consistent with enzyme-catalyzed attack of water at the ketone carbonyl. 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 this enzyme may have an alternative function, for example, as a base.