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

2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) fr om Escherichia coli has been purified to near homogeneity from an over expressing strain of E. coli. The purified enzyme is a 29 kDa dimeric protein requiring no cofactors for catalytic activity. The enzyme has a K-m of 2.1 mu M and a k(cat) of 36 s(-1) for its natural substrate a nd shows high selectivity for the propionate side chain of the substra te. The stereochemical course of the MhpC reaction was elucidated by c onversion of protiosubstrate in (H2O)-H-2 and conversion of deuteriate d substrate in (H2O)-H-1, revealing that the reaction proceeds with ov erall replacement of a succinyl moiety by a proton from water in the H -5(E) position, with retention of regiochemistry. Isotope exchange was also observed in the H-5(Z) position of the product, which was ration alized by enzyme-catalyzed exchange of H-2 into C-5 of the substrate f rom (H2O)-H-2. These data are consistent with a reversible keto-enol t automerization taking place as the first step of the enzyme mechanism.