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

Abstract 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 P450BM-3, P450terp, and P450cam, 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 Leu210 altered the responsiveness to the effector α-naphthoflavone and the regioselectivity of testosterone hydroxylation. Replacement of Leu211 by Ala also decreased the stimulation by α-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 α-naphthoflavone concentrations. Further characterization was performed with the additional effectors β-naphthoflavone, flavone, and 4-chromanone. The finding that P450 3A4 with one altered residue, Leu210 → 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.