Lipid regulation of bovine cytochrome P450(11) beta activity.
Purified bovine adrenocortical cytochrome P450(11) beta 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(11) beta exhibits a completely different response to changes in the fatty acyl groups from that shown by cytochrome P450scc. Cytochrome P450(11) beta 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 11 beta hydroxylase activity; most of this kinetic effect is attributable to a significant decrease in Vmax accompanied by modest changes in Km for the steroid substrate deoxycorticosterone. Diphosphatidyl glycerol (cardiolipin), which has been previously shown to activate cytochrome P450scc, is a potent inhibitor of the 11 beta hydroxylase activity of cytochrome P450(11) beta, 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 Vmax and relatively large increases (up to sevenfold) in Km for the steroid substrate. These effects on the 11 beta hydroxylase activity may have important implications for the in vivo regulation of not only the 11 beta hydroxylase activity, but also the other catalytic activities of this enzyme, particularly 18- and 19-hydroxylase and oxidase activities.
Seybert
D W
DW
Department of Chemistry, Duquesne University, Pittsburgh, Pennsylvania 15282.
D000311:Adrenal Glands / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D003900:Desoxycorticosterone / Q000378:metabolism; D005227:Fatty Acids / Q000494:pharmacology; D007700:Kinetics; D008055:Lipids / Q000494:pharmacology*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011136:Polysorbates / Q000494:pharmacology; D013252:Steroid 11-beta-Hydroxylase / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D005227:Fatty Acids; D008055:Lipids; D010743:Phospholipids; D011136:Polysorbates; D003900:Desoxycorticosterone; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D013252:Steroid 11-beta-Hydroxylase