Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
Amino acid 305 determines catalytic center accessibility in CYP3A4
Autore:
Fowler, SM; Riley, RJ; Pritchard, MP; Sutcliffe, MJ; Friedberg, T; Wolf, CR;
Indirizzi:
AstraZeneca R&D Charnwood, Dept Phys & Metab Sci, Loughborough LE11 5RH, Leics, England AstraZeneca R&D Charnwood Loughborough Leics England LE11 5RH cs, England Ninewells Hosp & Med Sch, Ctr Biomed Res, Dundee DD1 9SY, Scotland Ninewells Hosp & Med Sch Dundee Scotland DD1 9SY undee DD1 9SY, Scotland Univ Leicester, Dept Chem, Leicester LE1 7RH, Leics, England Univ Leicester Leicester Leics England LE1 7RH er LE1 7RH, Leics, England
Titolo Testata:
BIOCHEMISTRY
fascicolo: 15, volume: 39, anno: 2000,
pagine: 4406 - 4414
SICI:
0006-2960(20000418)39:15<4406:AA3DCC>2.0.ZU;2-9
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN CYTOCHROME-P450 3A4; SITE-DIRECTED MUTAGENESIS; ESCHERICHIA-COLI; ACTIVE-SITE; OXYGEN ACTIVATION; P450 REDUCTASE; 2D6; CYTOCHROMES-P450; IDENTIFICATION; KETOCONAZOLE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
32
Recensione:
Indirizzi per estratti:
Indirizzo: Fowler, SM AstraZeneca R&D Charnwood, Dept Phys & Metab Sci, Bakewell Rd, Loughborough LE11 5RH, Leics, England AstraZeneca R&D Charnwood Bakewell RdLoughborough Leics England LE11 5RH
Citazione:
S.M. Fowler et al., "Amino acid 305 determines catalytic center accessibility in CYP3A4", BIOCHEM, 39(15), 2000, pp. 4406-4414

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 wildtype CYP3A4, The A305F mutation abolished diazepam oxidase activity and reduced theS-50 and V-max for erythromycin N-demethylase activity from 17 to 10 mu M and from 3.2 to 1.2 pmol product/min/pmol P350, respectively. The Vm, for testosterone 6 beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P350, whereas the S-50 increased from 33to 125 mu M. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S50 increased from 9 to 42 mu M. The K-i for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 mu M, from 0.052 to 0.73 mu M, and from 0.043 to 2.2 mu M by the A305F mutation when measuredagainst 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 6 beta-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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 22/01/20 alle ore 19:03:34