Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity

Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 si tes within CYP3A4 proposed to be involved in substrate specificity or coope rativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of and rostenedione. The function of the 25 mutants was assessed in a reconstitute d system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)c oumarin (7-BFC), and alpha -naphthoflavone (ANF) as substrates. CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC deb enzylation. Analysis of 12 mutants with significant steroid hydroxylase act ivity showed a lack of positive correlation between ANF oxidation and stimu lation of progesterone 6 beta -hydroxylation by ANF, indicating that ANF bi nds at two sites within CYP3A4. 7-BFC debenzylation was stimulated by proge sterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6 b eta -hydroxylation. Correlational analysis showed no relationship between 7 -BFC debenzylation and either progesterone or testosterone 6 beta -hydroxyl ation. These data are difficult to explain with a two-site model of CYP3A4 but suggest that three subpockets exist within the active site. Interesting ly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind a t preferred locations in the CYP3A4 binding pocket.