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
Dc. Swinney et Ay. Mak, 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, Biochemistry, 33(8), 1994, pp. 2185-2190
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.