Dual role of human cytochrome P450 3A4 residue phe-304 in substrate specificity and cooperativity

The structural basis of cooperativity of progesterone hydroxylation catalyz ed by human cytochrome P450 3A4 has been investigated. A recent study sugge sted that substitution of larger side chains at positions Leu-211 and Asp-2 14 partially mimics the action of effector by reducing the size of the acti ve site. Based on predictions from molecular modeling that Phe-304 in the h ighly conserved I helix is involved in both effector and substrate binding, a tryptophan residue was substituted at this position. The purified F304W mutant displayed hyperbolic progesterone hydroxylase kinetics, indicating a lack of homotropic cooperativity. However, the mutant remained responsive to stimulation by alpha-naphthoflavone, exhibiting a 2-fold decrease in the K-m value for progesterone 6 beta-hydroxylation in the presence of 25 mu M effector. Combining substitutions to yield the triple mutant L211F/D214E/F 304W maintained the V-max and decreased the K-m for progesterone 6 beta-hyd roxylation, minimized stimulation by alpha-naphthoflavone, and decreased th e rate of alpha-naphthoflavone oxidation to one-eighth of the wild type. In terestingly, the Delta A(max) for spectral binding of alpha-naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that proge sterone and alpha-naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket, although both substrates appear to have equal access to the reactive oxygen.