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Titolo:
Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity
Autore:
Domanski, TL; He, YA; Khan, KK; Roussel, F; Wang, QM; Halpert, JR;
Indirizzi:
Univ Texas, Med Branch, Dept Pharmacol & Toxicol, Galveston, TX 77555 USA Univ Texas Galveston TX USA 77555 acol & Toxicol, Galveston, TX 77555 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 34, volume: 40, anno: 2001,
pagine: 10150 - 10160
SICI:
0006-2960(20010828)40:34<10150:PATSMO>2.0.ZU;2-W
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN CYTOCHROME-P450 3A4; HUMAN-LIVER; DIRECTED MUTAGENESIS; ALPHA-NAPHTHOFLAVONE; BINDING SITES; ACTIVE-SITE; ACTIVATION; METABOLISM; KINETICS; HYDROXYLATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
44
Recensione:
Indirizzi per estratti:
Indirizzo: Domanski, TL Univ Texas, Med Branch, Dept Pharmacol & Toxicol, 301 Univ Blvd, Galveston, TX 77555 USA Univ Texas 301 Univ Blvd Galveston TX USA 77555 TX 77555 USA
Citazione:
T.L. Domanski et al., "Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity", BIOCHEM, 40(34), 2001, pp. 10150-10160

Abstract

Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from acomparison with the structure of P450(eryF) containing two molecules of androstenedione. The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and alpha -naphthoflavone (ANF) as substrates. CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation. Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6 beta -hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A4. 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6 beta -hydroxylation. Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6 beta -hydroxylation. These data are difficult to explain with a two-site model of CYP3A4but suggest that three subpockets exist within the active site. Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A4 binding pocket.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 29/02/20 alle ore 14:35:22