title: The influence of the amino acid substitution I98K on the catalytic activity of baboon cytochrome P450 side-chain cleavage (CYP11A1).
abstract: Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the first and "rate-limiting" step in steroidogenesis, the conversion of cholesterol to pregnenolone. In an effort to gain further insight into the structure/function relationship of this key enzyme, CYP11A1 was characterized in the Cape baboon (Papio ursinus), a species closely related to humans. Baboon cDNA was isolated from adrenal tissue and direct sequence analysis showed mature baboon and human CYP11A1 share 98% deduced amino acid homology. The cDNA was subsequently amplified and two recombinant constructs, CYP11A1a and CYP11A1b, were cloned. Sequence analyses of the constructs revealed four amino acid substitutions. The constructs were expressed in nonsteroidogenic mammalian COS-1 cells with 25-hydroxycholesterol as substrate. Apparent Km values of 1.62 and 4.53 microM were determined for CYP11A1a and CYP11A1b, respectively. Homology modeling revealed that the lower substrate affinity of CYP11B1b could be attributed to an I98K substitution, which lies between the B and C helices, providing further evidence for the importance of this domain in the catalytic activity of CYP11A1.
authors.affiliation: Department of Biochemistry, University of Stellenbosch, Stellenbosch, South Africa.
authors.forename: K H
authors.initials: KH
authors.lastname: Storbeck
authors.forename: P
authors.initials: P
authors.lastname: Swart
authors.forename: S
authors.initials: S
authors.lastname: Graham
authors.forename: A C
authors.initials: AC
authors.lastname: Swart
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000378:metabolism*; D007532:Isoleucine; D008239:Lysine; D008958:Models, Molecular; D008968:Molecular Conformation; D010215:Papio / Q000378:metabolism*; D014162:Transfection
chemical_list: D007532:Isoleucine; D002786:Cholesterol Side-Chain Cleavage Enzyme; D008239:Lysine