title: Amino acid 305 determines catalytic center accessibility in CYP3A4.
abstract: Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P450, whereas the S(50) increased from 33 to 125 microM. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S(50) increased from 9 to 42 microM. The K(i) for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 microM, from 0.052 to 0.73 microM, and from 0.043 to 2.2 microM by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively. Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6beta-hydroxylase (V(max) = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V(max) = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V(max) = 4.6 pmol product/min/pmol P450). K(i) values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.
authors.affiliation: Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, U.K. stephen.fowler@astrazeneca.com
authors.forename: S M
authors.initials: SM
authors.lastname: Fowler
authors.forename: R J
authors.initials: RJ
authors.lastname: Riley
authors.forename: M P
authors.initials: MP
authors.lastname: Pritchard
authors.forename: M J
authors.initials: MJ
authors.lastname: Sutcliffe
authors.forename: T
authors.initials: T
authors.lastname: Friedberg
authors.forename: C R
authors.initials: CR
authors.lastname: Wolf
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics*; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D003022:Clotrimazole / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003975:Diazepam / Q000378:metabolism; D004464:Econazole / Q000494:pharmacology; D004917:Erythromycin / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007654:Ketoconazole / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008825:Miconazole / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009543:Nifedipine / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D007093:Imidazoles; D009097:Multienzyme Complexes; D013739:Testosterone; D004917:Erythromycin; D004464:Econazole; D008825:Miconazole; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; C113764:Cyp2c13 protein, rat; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C118840:diazepam C3-hydroxylase; D010089:Oxidoreductases, N-Demethylating; C067539:diazepam N-demethylase; D003022:Clotrimazole; D009543:Nifedipine; D000409:Alanine; D003975:Diazepam; D007654:Ketoconazole