Characterization of the Steroid-Metabolizing Capacity of the Hepatic Cytochrome P450IIC5 Expressed in COS-1 Cells: 3β-Hydroxysteroid Dehydrogenase/Δ5→ 4Isomerase Type Activity

Cytochrome P450IIC5 (rabbit liver 21-hydroxylase) is unusual among hepatic forms of cytochromes P450 because it catalyzes the conversion of one active steroid hormone (progesterone) to another active hormone (deoxycorticosterone). Another interesting aspect of this steroidhydroxylating enzyme is the ability to convert Δ5-3β-hydroxysteroids to the Δ4-3-ketosteroid configuration. The Δ5-3β-hydroxysteroid, pregnenolone, was readily 21-hydroxylated, and this product was further metabolized to the Δ4-3-ketosteroid, deoxycorticosterone. It is suggested that the mechanism of this cytochrome P450-mediated, 3β-hydroxysteroid dehydrogenase/Δ5 → 4isomerase-like reaction is through a gem-diol formation. In this study, COS-1 cells were transfected with the plasmid encoding cytochrome P450IIC5 to express a functional enzyme within the cell milieu. Transfected COS cells preferentially metabolize pregnenolone compared with all other steroids tested. Progesterone and 17α-hydroxypregnenolone are also 21-hydroxylated, whereas 17α-hydroxyprogesterone is a poor substrate. Substrate preference of this 21-hydroxylase differs from that seen with bovine adrenal P450XXIA1 (formerly P450C21) hydroxylase. Additionally, this study demonstrated that C19steroids, like dehydroepiandrosterone and androstenedione, are hydroxylated at the 16α position. Contrary to previous reports, no metabolite of estradiol-17β was detected, presumably due to the unstable nature of catechol estrogens (2-hydroyestradiol).