P450c17 mutations R347H and R358Q selectively disrupt 17,20-lyase activityby disrupting interactions with P450 oxidoreductase and cytochrome b(5)

Cytochrome P450c17 catalyzes steroid 17 alpha-hydroxylase and 17,20-lyase a ctivities and hence is a key enzyme in the production of human glucocortico ids and sex steroids. These two activities are catalyzed in a single substr ate-binding site but are regulated independently in human physiology. We ha ve recently shown that cytochrome b(5) facilitates 17,20-lyase activity by allosterically promoting the interaction of P450c17 with P450 oxidoreductas e (OR) and that the human P450c17 mutations, R347H and R358Q, selectively d estroy 17,20-lyase activity while sparing 17 alpha-hydroxylase activity. We transfected COS-1 cells with vectors for these P450c17 mutants and found t hat an excess of OR and b(5) restored a small amount of 17,20-lyase activit y, suggesting the mutations interfere with electron donation. To determine whether these mutations selectively interfere with the interaction of P450c 17 and its electron-donating system, we expressed each P450c17 mutant in ye ast with or without OR, b(5), or both, and measured enzyme kinetics in yeas t microsomes using pregnenolone and 17 alpha-hydroxypregnenolone as substra tes. The apparent Michaelis-Menten (K-m) values for the R347H mutant with a nd without coexpressed OR were 0.2 and 0.6 mu M, respectively, and for the R358Q mutant with and without OR they were 0.3 and 0.4 mu M, respectively; these values did not differ significantly from the wild-type values of 0.4 and 0.8 mu M with and without OR, respectively. Furthermore, coincubation w ith 17 alpha-hydroxypregnenolone showed a competitive mechanism for interfe rence of catalysis. The similar kinetics and the competitive inhibition pro ve that the mutations did not affect the active site. Coexpression of the m utants with OR yielded insignificant 17,20-lyase activity, but addition of a 30:1 molar excess cytochrome b(5) to these microsomes restored partial 17 ,20-lyase activity, with the R358Q mutant achieving twice the activity of t he R347H mutant. These data indicate that both mutations selectively interf ere with 17,20-lyase activity by altering the interaction of P450c17 with O R, thus proving that the lyase activity was disrupted by interfering with e lectron transfer. Furthermore, the data offer the first evidence that R347 is a crucial component of the site at which b(5) interacts with the P450c17 .OR complex to promote electron transfer.