Catalytic properties of cytochrome P-450 scc purified from the human placenta: comparison to bovine cytochrome P-450 scc

Abstract

Cytochrome P-450 scc was purified from the human placenta by extraction of mitochondria with cholate and Emulgen 911, chromatography on phenyl-Sepharose and DEAE-Sephacel, and ammonium sulphate fractionation. The catalytic properties of the purified human cytochrome P-450 scc were analysed in Tween-20 micelles and compared to those of bovine adrenal cytochrome P-450 scc analysed in the same system. Both enzymes had the same K m for cholesterol and were stimulated by cardiolipin when the cholesterol concentration was subsaturating. Examination of the rates of pregnenolone synthesis from 20α-hydroxycholesterol, 22R-hydroxycholesterol and 20α,22R-dihydroxycholesterol by human and bovine cytochromes P-450 scc revealed that the first hydroxylation (22R position) was rate-limiting for both in Tween-20 micelles. The rate of the 22R-hydroxylation was further decreased when a 20α-hydroxyl group was already present on the cholesterol side-chain. The second hydroxylation occurred at about the same rate as the third hydroxylation for both enzymes. The rate of side-chain cleavage of 25-hydroxycholesterol by human cytochrome P-450 scc in Tween-20 micelles was low, the highest rate being about 1% of the Vm max for cholesterol. Substrate inhibition was seen with high concentrations of 25-hydroxycholesterol. Conversion of 25-hydroxycholesterol to pregnenolone was accompanied by a build-up of products with intact side-chains, which were probably intermediates of the reaction. Side-chain cleavage of 25-hydroxycholesterol by bovine cytochrome P-450 scc showed similar characteristics to the human enzyme, except that the highest velocity observed was approx. 25% of the V max for cholesterol. Rates of cleavage of 25-hydroxycholesterol by both enzymes were higher in dioleoylphosphatidylcholine vesicles than in Tween-20, but were still well below the V max for cholesterol and showed substrate inhibition. This study shows that there is close similarity in catalytic properties between human and bovine cytochromes P-450 scc which suggests that the active site of the cytochrome is highly conserved.