Send to

Choose Destination
Biochemistry. 1997 Oct 7;36(40):12242-51.

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.

Author information

Department of Chemistry, University of Southampton, Highfield, U.K.


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 microM 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.

[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center