title: The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities.
abstract: CYP17 (17alpha-hydroxylase-17,20-lyase; also P450c17 or P450(17alpha)) catalyses the17alpha-hydroxylation of progestogens and the subsequent acyl-carbon cleavage of the 17alpha-hydroxylated products (lyase activity) in the biosynthesis of androgens. The enzyme also catalyses another type of acyl-carbon cleavage (direct cleavage activity) in which the 17alpha-hydroxylation reaction is by-passed. Human CYP17 is heavily dependent on the presence of the membrane form of cytochrome b(5) for both its lyase and direct cleavage activities. In the present study it was found that substitution of human CYP17 amino acids, Arg(347), Arg(358) and Arg(449), with non-cationic residues, yielded variants that were impaired in the two acyl-carbon bond cleavage activities, quantitatively to the same extent and these were reduced to between 3 and 4% of the wild-type protein. When the arginines were replaced by lysines, the sensitivity to cytochrome b(5) was restored and the acyl-carbon cleavage activities were recovered. All of the human mutant CYP17 proteins displayed wild-type hydroxylase activity, in the absence of cytochrome b(5). The results suggest that the bifurcated cationic charges at Arg(347), Arg(358) and Arg(449) make important contributions to the formation of catalytically competent CYP17.cytochrome b(5) complex. The results support our original proposal that the main role of cytochrome b(5) is to promote protein conformational changes which allow the iron-peroxo anion to form a tetrahedral adduct that fragments to produce the acyl-carbon cleavage products.
authors.affiliation: Department of Chemistry, Centre for Chemical Biology, University of Sheffield, Sheffield S3 7HF, UK. p.lee-robichaud@sheffield.ac.uk
authors.forename: Peter
authors.initials: P
authors.lastname: Lee-Robichaud
authors.forename: Monika E
authors.initials: ME
authors.lastname: Akhtar
authors.forename: J Neville
authors.initials: JN
authors.lastname: Wright
authors.forename: Qaiser I
authors.initials: QI
authors.lastname: Sheikh
authors.forename: Muhammad
authors.initials: M
authors.lastname: Akhtar
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013254:Steroid 17-alpha-Hydroxylase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D015786:Cytochromes b5; D001120:Arginine; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase