MODULATION OF 17-ALPHA-HYDROXYLASE 17,20-LYASE ACTIVITY OF GUINEA-PIGCYTOCHROME P450C17 BY SITE-DIRECTED MUTAGENESIS/

Microsomal cytochrome P450c17 (17 alpha-hydroxylase/l7,20-lyase) catal yzes two reactions in the Delta(5) and Delta(4) pathways leading to th e production of C-19 steroids. Transient expression of human, bovine, porcine, rat, and mouse p450c17 cDNAs showed that the protein has 17 a lpha-hydroxylase and 17,20-lyase activities, converting pregnenolone a nd progesterone into Delta(5)- and Delta(4)-C-19 steroids, respectivel y, although the rat and mouse proteins have a preferential pathway tow ard the Delta(4) steroids. The guinea pig (gp) P450c17 shares 46% to 7 0% amino acid identity with the corresponding proteins of other specie s, and further characterization indicated that the guinea pig enzyme o nly converts progesterone to androstenedione, In this study, we have t ried to identify amino acid(s) in the gpP450c17 that governs such a st eroid specificity. Among the various mutants that we have created, cha nge of the arginine (R) residue at position 200 to an asparagine (N) ( R200N) in the gpP450c17 protein increased reactivity toward pregnenolo ne compared with the mild-type enzyme. Pregnenolone was converted into 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. However, thi s gain occurred at the expense of the 17,20-lyase activity toward 17 a lpha-hydroxyprogesterone. The R200N mutation in the gpP350c17 protein introduced a potential N-linked glycosylation site ((200)Asn-X-Thr(202 )); however, substitution of the Thr(202) residue by an asparagine (R2 00N/T202N), which abolishes the site, did not change the preference of the gpP350c17 mutant for pregnenolone, Furthermore, introduction of a putative glycosylation site at amino acid 185 in the gpP350c17 enzyme did not alter substrate specificity. The properties of the amino acid were also investigated, and neither the charge nor the size of the si dechain elicited change in the substrate specificity of gpP350c17. Thu s, our results demonstrate that the mutation of arginine to asparagine at position 200 changes the substrate specificity of the gpP450c17 en zyme.