A MODEL FOR HUMAN CYTOCHROME-P-450 2D6 BASED ON HOMOLOGY MODELING ANDNMR-STUDIES OF SUBSTRATE-BINDING

The cytochrome P-450 responsible for the debrisoquine/sparteine polymo rphism (P-450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was inco rporated at the C-terminus of the expressed protein, which, after puri fication of the protein on a nickel-agarose column, could be removed p roteolytically by treatment with thrombin. Purified yields of P-450 2D 6 were 2.4 mg from 700 mt of cell culture, The protein had a greater t han 90% heme content and was fully active, having no residual absorban ce at 420 nm in the reduced CO complex. The quantities produced allowe d direct study of the interaction of the substrate codeine with the en zyme by paramagnetic relaxation effects on the NMR spectrum of the sub strate. Distances between the heme iron atom and substrate protons wer e calculated from these experiments, and the orientation of the substr ate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 Angstrom), consistent wit h the observed O-demethylation to morphine. A model of the complex of P-450 2D6 with codeine was built from a multiple sequence and structur e alignment of the known crystal structures for P(450)s, incorporating the experimental constraints derived from the NMR studies. This showe d that the overall fold of P-450 2D6 is more similar to that of P-450 BM3 than to either P-450 cam or P-450 terp. Codeine binds to P-450 2D6 so that the methoxy group is directly above the A ring of the heme, w hile the basic nitrogen interacts with the carboxylate of aspartate 30 1.