Open AccessHuman cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase.
Author(s) -
Manjunath S. Shet,
Charles Fisher,
P L Holmans,
Ronald W. Estabrook
Publication year - 1993
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.90.24.11748
Subject(s) - benzphetamine , cytochrome b5 , biochemistry , cytochrome p450 , hydroxylation , reductase , chemistry , enzyme , biology
Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids; such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5 for the 6 beta-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.