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 (17α-hydroxylase-17,20-lyase; also P450c17 or P450 17α ) catalyses the17α-hydroxylation of progestogens and the subsequent acyl-carbon cleavage of the 17α-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 17α-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.