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 chemic als, including the immunosuppressive agent cyclosporin; macrolide anti biotics, 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 r eductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzym atically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expr essed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochr ome b5 for the 6beta-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-de methylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabo lism of triacetyloleandomycin, and this has a pronounced inhibitory ef fect on the metabolism of both testosterone and erythromycin. These re sults relate to the interpretation of current methods used to assess t he in vivo activity of P450 3A4.