Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase.
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.
Shet
M S
MS
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
Fisher
C W
CW
Holmans
P L
PL
Estabrook
R W
RW
D001483:Base Sequence; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System; D015786:Cytochromes b5 / Q000378:metabolism; D017931:DNA Primers; D004917:Erythromycin / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D009543:Nifedipine / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013739:Testosterone; D004917:Erythromycin; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D009251:NADPH-Ferrihemoprotein Reductase; D009543:Nifedipine