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.
Woods
S T
ST
Department of Biochemistry, The University of Western Australia, Nedlands, Australia.
Sadleir
J
J
Downs
T
T
Triantopoulos
T
T
Headlam
M J
MJ
Tuckey
R C
RC
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli; D005260:Female; D006801:Humans; D007532:Isoleucine; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D007532:Isoleucine; D002786:Cholesterol Side-Chain Cleavage Enzyme