Lipid regulation of bovine cytochrome P450 11β activity

Abstract

Purified bovine adrenocortical cytochrome P450 11β has been reconstituted into phospholipid vesicles using a detergent dialysis procedure. Using this reconstituted system, we have examined the effect of changes in the fatty acyl substituents of the lipids on the catalytic activity of the enzyme. The studies reported here show that cytochrome P450 11β exhibits a completely different response to changes in the fatty acyl groups from that shown by cytochrome P450 scc. Cytochrome P450 11β displays maximal activity in lipid vesicles composed of saturated lipids, such as dipalmitoyl and dimyristoyl phosphatidylcholines, with turnover numbers ranging from 35 to 60 min −1. Incremental increases of phospholipids such as diphytanoyl and dioleoyl phosphatidylcholines result in a progressive inhibition of 11β hydroxylase activity; most of this kinetic effect is attributable to a significant decrease in V max accompanied by modest changes in K m for the steroid substrate deoxycorticosterone. Diphosphatidyl glycerol (cardiolipin), which has been previously shown to activate cytochrome P450 scc, is a potent inhibitor of the 11β hydroxylase activity of cytochrome P450 11β, with half maximum inhibition observed in vesicles containing 4–5 mol% diphosphatidyl glycerol. Kinetic analysis demonstrates that this inhibition by diphosphatidyl glycerol is reflected in both a decrease in V max and relatively large increases (up to sevenfold) in K m for the steroid substrate. These effects on the 11β hydroxylase activity may have important implications for the in vivo regulation of not only the 11β hydroxylase activity, but also the other catalytic activities of this enzyme, particularly 18- and 19-hydroxylase and oxidase activities.