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 heroin metabolites and a biotransformation process for production of hydromorphone and hydrocodone. To improve these processes we have undertaken a thorough examination of the kinetic mechanism of MDH. Sequence comparisons indicated 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 kinetic studies, product inhibition patterns suggested that MDH follows a Theorell-Chance mechanism for codeinone reduction at pH 7, and a non-Theorell-Chance sequential ordered mechanism for codeine oxidation at pH 9.5. Residues corresponding to the catalytically important Tyr-48, Lys-77 and Asp-43 of aldose reductase were modified by site-directed mutagenesis, resulting in substantial loss of activity consistent with a catalytic role for these residues. Loss of activity of MDH in the presence of the reaction product morphinone 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.
Walker
E H
EH
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, U.K.
French
C E
CE
Rathbone
D A
DA
Bruce
N C
NC
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D004789:Enzyme Activation; D004795:Enzyme Stability; D004091:Hydromorphone / Q000031:analogs & derivatives / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D013379:Substrate Specificity
C036083:morphinone; D000429:Alcohol Oxidoreductases; C045221:morphine 6-dehydrogenase; D004091:Hydromorphone