Enzymatic properties of vesicle-reconstituted human cytochrome P450SCC (CYP11A1) differences in functioning of the mitochondrial electron-transfer chain using human and bovine adrenodoxin and activation by cardiolipin.

The recently reported heterologous expression and purification of both human cytochrome P450SCC and adrenodoxin [Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Haedlam, M.J. & Tuckey, R.C. (1998) Arch. Biochem. Biophys. 353, 109-115] has enabled us to perform studies with the membrane-reconstituted human enzymes to better understand the side-chain cleavage reaction in humans. Human P450SCC was successfully reconstituted into dioleoylphosphatidylcholine vesicles with and without cardiolipin and its enzymatic properties characterized in the membrane-bound state. Enhancement of the P450SCC activity and significant activation by cardiolipin were observed when human adrenodoxin instead of bovine adrenodoxin was used as electron donor. In the absence of cardiolipin, Km for cholesterol was decreased twice in the case of human adrenodoxin indicating enhanced cholesterol binding. On the other hand, in the presence of cardiolipin in the membrane both Km and V for cholesterol were decreased with human adrenodoxin as electron donor. Kinetic analysis of the interaction between human P450SCC and its redox partners provided evidence for enhanced binding of the human electron donor to human P450SCC indicated by both an increased V and decreased Kd for human adrenodoxin compared with the values with bovine adrenodoxin. Because no similar effects were observed in Tween 20 micelles, these results suggest that the phospholipid membrane may play an important role in the interaction of human adrenodoxin with human P450SCC.

Expression of catalytically active human cytochrome p450scc in Escherichia coli and mutagenesis of isoleucine-462.

Cytochrome P450scc (P450scc) catalyzes the first step in steroid hormone synthesis, the conversion of cholesterol to pregnenolone. Human P450scc has been poorly studied due to the difficulty of purifying reasonable quantities of enzyme from human tissue. To provide a more convenient source of the human enzyme and to enable structure-function studies to be done using site-directed mutagenesis, we expressed the mature form of human P450scc in Escherichia coli. The expression system enabled us to produce larger quantities of active cytochrome than have previously been isolated from placental mitochondria. The expressed P450scc was purified to near homogeneity and shown to have catalytic properties comparable to the enzyme purified from the human placenta. The mature form of human adrenodoxin was also expressed in E. coli and supported cholesterol side chain cleavage activity with the same Vmax as that observed using bovine adrenodoxin but with a higher Km. Mutation of Ile-462 to Leu in human P450scc caused a decrease in the catalytic rate constant (kcat) with cholesterol as substrate, increased the Km for 22R-hydroxycholesterol, but did not affect the kinetic constants for 20 alpha-hydroxycholesterol. This suggests that Ile-462 lies close to the side chain binding site and that the side chains of cholesterol, 22R-hydroxycholesterol, and 20 alpha-hydroxycholesterol occupy slightly different positions in the active site.

Electron transfer to cytochrome P-450scc limits cholesterol-side-chain-cleavage activity in the human placenta.

The aim of this study was to determine whether electron transfer from adrenodoxin reductase and adrenodoxin limits the activity of cytochrome P-450scc in mitochondria from the human placenta. Mitochondria were disrupted by sonication to enable exogenous adrenodoxin and adrenodoxin reductase to deliver electrons to cytochrome P-450scc. After sonication, the rate of pregnenolone synthesis was greatly decreased relative to that by intact mitochondria, due to dilution of endogenous adrenodoxin and adrenodoxin reductase into the incubation medium. The addition of saturating concentrations of bovine or human adrenodoxin and bovine adrenodoxin reductase to the disrupted mitochondria gave an initial rate of pregnenolone synthesis that was 6.3-fold higher than that for intact mitochondria. Similar results were observed when 20alpha-hydroxycholesterol was used as substrate rather than endogenous cholesterol. The turnover number of cytochrome P-450scc in sonicated placental mitochondria supplemented with adrenodoxin and adrenodoxin reductase was comparable to that for the purified enzyme assayed under conditions where electron transfer was not limiting. Addition of exogenous adrenodoxin and adrenodoxin reductase to sonicated mitochondria from the pig corpus luteum and rat adrenal had a much smaller effect on pregnenolone synthesis compared with intact mitochondria, than observed for the placenta. We conclude that in the human placenta, electron transfer to cytochrome P-450scc is limiting, permitting pregnenolone synthesis to proceed at only 16% maximum velocity.