A review of mechanistic studies on aromatase (CYP19) and 17α-hydroxylase-17,20-lyase (CYP17).

In the conventional P-450 dependent hydroxylation reaction, the Fe(III) resting state of the enzyme, by a single electron transfer, is reduced to Fe(II), which reacts with O(2) to produce a Fe(III)-O-O intermediate. The latter following the transfer of another electron furnishes a ferric-peroxyanion, Fe(III)-O-O(-), which after protonation leads to the fission of the O-O bond resulting in the formation of Fe(V)O, the key player in the hydroxylation process. Certain members of the P-450 family, including CYP17 and CYP19, catalyze, at the same active site, not only the hydroxylation process but also an acyl-carbon bond cleavage reaction which has been interpreted to involve the nucleophilic attack of the ferric-peroxyanion, Fe(III)-O-O(-), on the acyl carbon to furnish a tetrahedral intermediate which fragments, leading to acyl-carbon cleavage. Evidence is presented to show that in the case of CYP17 the attack of Fe(III)-O-O(-) on the target carbon is promoted by cytochrome b(5), which acts as a conformational regulator of CYP17. It is this regulation of CYP17 that provides a safety mechanism which ensures that during corticoid biosynthesis, which involves 17α-hydroxylation by CYP17, androgen formation is avoided. Finally, a brief account is presented of the inhibitors, of the two enzymes, which have been designed on the basis of their mechanism of action. Article from the Special issue on 'Targeted Inhibitors'.

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.

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.