ALANINE-SCANNING MUTAGENESIS OF A PUTATIVE SUBSTRATE RECOGNITION SITEIN HUMAN CYTOCHROME-P450 3A4 - ROLE OF RESIDUE-210 AND RESIDUE-211 INFLAVONOID 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 huma n liver. Mutagenesis of this region, which has been proposed to align with the C-terminal ends of F-helices from cytochromes P450(BM-3), P45 0(terp), and P450(cam), served as a test of the applicability of the s ubstrate recognition site model of Gotoh (Gotoh, O. (1992) J. Biol. Ch em. 267, 83-90) to P450 3A4. The results, using two steroid substrates , indicated that substitution of Ala for Leu(210) altered the responsi veness to the effector alpha-naphthoflavone and the regioselectivity o f testosterone hydroxylation, Replacement of Leu(211) by Ala also decr eased the stimulation by alpha-naphthoflavone, whereas mutations at re sidues 212-216 had little effect, The diminished flavonoid responses o f the 210 and 211 mutants were observed over a wide range of progester one and alpha-naphthoflavone concentrations. Further characterization was performed with the additional effectors beta-naphthoflavone, flavo ne, and 4-chromanone. The finding that P450 3A4 with one altered resid ue, Leu(210) --> Ala, can have both an altered testosterone hydroxylat ion profile and response to flavonoid stimulation provides evidence th at the substrate binding and effector sites are at least partially ove rlapping.