EXPRESSION OF CATALYTICALLY ACTIVE HUMAN CYTOCHROME P450SCC IN ESCHERICHIA-COLI AND MUTAGENESIS OF ISOLEUCINE-462

Cytochrome P450scc (P450scc) catalyzes the first step in steroid hormo ne synthesis, the conversion of cholesterol to pregnenolone, Human P45 0scc has been poorly studied due to the difficulty of purifying reason able quantities of enzyme from human tissue. To provide a more conveni ent source of the human enzyme and to enable structure-function studie s to be done using site-directed mutagenesis, we expressed the mature form of human P450scc in Escherichia coli. The expression system enabl ed us to produce larger quantities of active cytochrome than have prev iously been isolated from placental mitochondria, The expressed P450sc c was purified to near homogeneity and shown to have catalytic propert ies comparable to the enzyme purified from the human placenta. The mat ure form of human adrenodoxin was also expressed in E. coli and suppor ted cholesterol side chain cleavage activity with the same V-max as th at observed using bovine adrenodoxin but with a higher K-m. Mutation o f Ile-462 to Leu in human P450scc caused a decrease in the catalytic r ate constant (k(cat)) with cholesterol as substrate, increased the K-m 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 cholestero l, 22R-hydroxycholesterol, and 20 alpha-hydroxycholesterol occupy slig htly different positions in the active site. (C) 1998 Academic Press.