Active site models of human cytochrome P450s based on NMR relaxation measurements

The various mammalian cytochrome P450s (2D6, 3A4. etc.) have been produced in large quantities by expression of their cDN As in baculovirus or e.coli. A poly-histidine extension has been incorporated at the C-terminus of the expressed proteins, which, after purification of the protein on a nickelagarose column, could be removed proteolytically by treatment with thrombin. The quantities produced allowed direct study of the interaction of substrates with P450s using NMR. We have studied the binding of the different substrates to the Cytochrome P450 2D6 and 3 A4 by measurements of the relaxation effects of the unpaired electron of the haem iron on the protons of the bound substrates'. Using these measurements on the substrate protons and the known X-ray crystal structures of four P450s, a model for the initial enzyme-substrate complex was built. Based on these results, mutants were designed to explore the substrate specificity of the enzyme. In Cytochrome P450 2D6, the F483I mutant was shown to accommodate larger substrate (like testosterone) as compared to wild type2.