Purification, Characterization, and Stereochemical Analysis of a C−C Hydrolase: 2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic Acid 5,6-Hydrolase
- 1 October 1997
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 36 (40) , 12242-12251
- https://doi.org/10.1021/bi971115r
Abstract
2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli has been purified to near homogeneity from an overexpressing strain of E. coli. The purified enzyme is a 29 kDa dimeric protein requiring no cofactors for catalytic activity. The enzyme has a Km of 2.1 μM and a kcat of 36 s-1 for its natural substrate and shows high selectivity for the propionate side chain of the substrate. The stereochemical course of the MhpC reaction was elucidated by conversion of protiosubstrate in 2H2O and conversion of deuteriated substrate in 1H2O, revealing that the reaction proceeds with overall replacement of a succinyl moiety by a proton from water in the H-5E position, with retention of regiochemistry. Isotope exchange was also observed in the H-5Z position of the product, which was rationalized by enzyme-catalyzed exchange of 2H into C-5 of the substrate from 2H2O. These data are consistent with a reversible keto-enol tautomerization taking place as the first step of the enzyme mechanism.Keywords
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