Alanine-scanning mutagenesis of a putative substrate recognition site in human cytochrome P450 3A4 - Role of residues 210 and 211 in flavonoid activation and substrate specificity

Alanine-scanning mutagenesis was performed on amino acid residues 210-216 of cytochrome P450 3A4, the major drug-metabolizing enzyme of human liver. Mutagenesis of this region, which has been proposed to align with the C-terminal ends of F-helices from cytochromes P450(BM-3), P450(terp), and P450(cam), served as a test of the applicability of the substrate recognition site model of Gotoh (Gotoh, O. (1992) J. Biol. Chem. 267, 83-90) to P450 3A4. The results, using two steroid substrates, indicated that substitution of Ala for Leu(210) altered the responsiveness to the effector alpha-naphthoflavone and the regioselectivity of testosterone hydroxylation, Replacement of Leu(211) by Ala also decreased the stimulation by alpha-naphthoflavone, whereas mutations at residues 212-216 had little effect, The diminished flavonoid responses of the 210 and 211 mutants were observed over a wide range of progesterone and alpha-naphthoflavone concentrations. Further characterization was performed with the additional effectors beta-naphthoflavone, flavone, and 4-chromanone. The finding that P450 3A4 with one altered residue, Leu(210) --> Ala, can have both an altered testosterone hydroxylation profile and response to flavonoid stimulation provides evidence that the substrate binding and effector sites are at least partially overlapping.