title: 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 alpha-naphthoflavone and the regioselectivity of testosterone hydroxylation. Replacement of Leu211 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, 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.
authors.affiliation: Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, USA. Harlow@tonic.pharm.arizona.edu
authors.forename: G R
authors.initials: GR
authors.lastname: Harlow
authors.forename: J R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000409:Alanine; D001189:Aryl Hydrocarbon Hydroxylases; D001345:Autoradiography; D001571:Benzoflavones / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D002867:Chromones / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D047309:Flavones; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011374:Progesterone / Q000378:metabolism; D012995:Solubility; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism; D013379:Substrate Specificity; D019324:beta-Naphthoflavone / Q000494:pharmacology
chemical_list: D001571:Benzoflavones; D002867:Chromones; D047309:Flavones; D005419:Flavonoids; C068514:4-chromanone; D011374:Progesterone; C011512:alpha-naphthoflavone; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C022724:testosterone 7-alpha-hydroxylase, hamster; D013254:Steroid 17-alpha-Hydroxylase; C022445:progesterone 17-alpha-hydroxylase; D000409:Alanine; C043562:flavone