Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Morphine dehydrogenase (MDH) of Pseudomonas putida M10 catalyses the NADP()-dependent oxidation of morphine and codeine to morphinone and codeinone. This enzyme forms the basis of a sensitive detection and assay method for h eroin metabolites and a biotransformation process for production of hydromo rphone and hydrocodone. To improve these processes we have undertaken a tho rough examination of the kinetic mechanism of MDH. Sequence comparisons ind icated that MDH belongs within the aldose reductase enzyme family. MDH was shown to be specific for the pro-R hydrogen of NADPH. In steady-state kinet ic studies, product inhibition patterns suggested that MDH follows a Theore ll-Chance mechanism for codeinone reduction at pH 7, and a non-Theorell-Cha nce sequential ordered mechanism for codeine oxidation at pH 9.5. Residues corresponding to the catalytically important Tyr-48, Lys-77 and Asp-43 of a ldose reductase were modified by site-directed mutagenesis, resulting in su bstantial loss of activity consistent with a catalytic role for these resid ues. Loss of activity of MDH in the presence of the reaction product morphi none was found to be due to the formation of a covalent adduct with Cys-80; alteration of Cys-80 to serine resulted in an enzyme with greatly enhanced stability.