ANDROGEN FORMATION BY CYTOCHROME-P450 CYP17 - SOLVENT ISOTOPE EFFECT AND PL STUDIES SUGGEST A ROLE FOR PROTONS IN THE REGULATION OF OXENE VERSUS PEROXIDE CHEMISTRY

CYP17 catalyzes the cleavage of the C-17 side chain of progesterone to form androstenedione. The two-step reaction involves an initial 17 al pha-hydroxylation catalyzed by oxene chemistry followed by cleavage of the C-17 side chain. We have recently shown that C-17 side-chain clea vage may involve the rearrangement of a peroxy intermediate via a Baey er-Villiger rearrangement [Mak, A. Y., and Swinney, D. C. (1992) J. Am . Chem. Sec, 114, 8309]. Accordingly, CYP17 is proposed to catalyze ox idations via both oxene and peroxide chemistry. This study was initiat ed to investigate the possibility that protons may play a determining role in differentiating between the oxene and peroxide chemistries ass ociated with product formation. The pL dependence of the deuterium sol vent isotope effects associated with progesterone oxidation to 17 alph a-hydroxyprogesterone and 17-O-acetyltestosterone and 17 alpha-hydroxy progesterone oxidation to androstenedione was determined in microsomes from pig testes. The formation of 17 alpha-hydroxyprogesterone is ass umed to occur via oxene chemistry and the formation of 17-O-acetyltest osterone and androstenedione by peroxide chemistry. The initial rate o f progesterone oxidation to 17 alpha-hydroxyprogesterone was associate d with a pL-independent inverse solvent isotope effect ((H)k/(D)k = 0. 75-0.95, in 30% DOD), whereas the rate of oxidation to 17-O-acetyltest osterone was associated with a pi-independent positive solvent isotope effect in the presence of 30% DOD ((H)k/(D)k approximately 2). In con trast, DOD inhibited the formation of androstenedione from 17 alpha-hy droxyprogesterone in a noncompetitive, pL-dependent manner. These data strongly suggest that protons do influence the chemistry of CYP 17 ca talysis. We interpret the pL-independent isotope effects to represent the effect of the fraction of deuterium upon protonation of the ferric peroxy intermediate (Fe-III-O-O-). DOD shifts the acid-base equilibri um to the protonated intermediate (Fe-III-O-O-H), increasing the rate of products formed via oxene chemistry at the expense of those formed from peroxide chemistry. This results in the inverse isotope effect ob served for 17 alpha-hydroxyprogesterone formation and the positive iso tope effect for 17-O-acetyltestosterone formation. The pL-dependent, n oncompetitive inhibition of androstenedione formation by DOD is interp reted to reflect the effect of shifting the pK of a rate-controlling i onization upon the concentration of a catalytically competent enzyme. We hypothesize that 17 alpha-hydroxyprogesterone binds to an unprotona ted form of the enzyme in a manner which interrupts a proton shuttle t o the ferric peroxy intermediate, thus facilitating the C-17 side-chai n cleavage via peroxide chemistry.