Properties of an adrenal cytochrome P-450 ( P-450 11 β) for the hydroxylations of corticosteroids

Abstract

A highly purified preparation of cytochrome P-450, designated as P-450 11 β , has been obtained from bovine adrenal cortex mitochondria. The P-450 11 β exhibits remarkably high steroid hydroxylase activity in the reconstituted adrenal electron-donating system from NADPH via NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1) and adrenal ferredoxin. The turnover numbers (moles of hydroxylated product formed per minute per mole of P-450-heme) are 110 and 18 for respective 11β- and 18-hydroxylase activity when deoxycorticosterone is the substrate. The apparent K m value is 6 μ m for both reactions. The ratio, about 6:1 between the two activities, is constant under various experimental conditions including those in the presence of competitive inhibitors of hydroxylation. In addition to deoxycorticosterone, other steroids such as 11-deoxycortisol, 4-androstene-3,17-dione and testosterone are the hydroxylatable substrates. In cases in which 4-androstene-3,17-dione, a C 19-steroid, is the substrate, the hydroxylatable sites appear to be its respective 11β- and 19-position. The ratio between the two activities is about 4:1. In view of these results, it is concluded that one hemoprotein species, the P-450 11 β , is responsible for the hydroxylase reactions of various Corticosteroids. 2-Methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) inhibits the P-450 11β-catalyzed steroid hydroxylase reactions of either deoxycorticosterone at 11β- and 18-position or 4-androstene-3,17-dione at 11β- and 19-position ( K i = 0.1-0.2 μM). The P-450 scc-catalyzed cholesterol desmolase reaction is also inhibited, although weakly ( K i = 160 μM). In addition, both adrenal cytochromes appeared to differ from each other in spectral response to metyrapone.