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Volume 272, Number 9, Issue of February 28, 1997 pp. 5396-5402
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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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

(Received for publication, October 22, 1996, and in revised form, December 23, 1996)

From the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721

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 Leu²¹⁰ altered the responsiveness to the effector -naphthoflavone and the regioselectivity of testosterone hydroxylation. Replacement of Leu²¹¹ by Ala also decreased the stimulation by -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 -naphthoflavone concentrations. Further characterization was performed with the additional effectors -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.

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