Rj. Auchus et Wl. Miller, Molecular modeling of human P450c17 (17 alpha-hydroxylase/17,20-lyase): Insights into reaction mechanisms and effects of mutations, MOL ENDOCR, 13(7), 1999, pp. 1169-1182
P450c17 (17 alpha-hydroxylase/17,20-lyase) catalyzes steroid 17 alpha-hydro
xylase and 17,20-lyase activities in the biosynthesis of androgens and estr
ogens. These two activities are differentially regulated in a tissue-specif
ic and developmentally programmed manner. To visualize the active site topo
logy of human P450c17 and to study the structural basis of its substrate sp
ecificity and catalytic selectivity, we constructed a second-generation com
puter-graphic model of human P450c17, The energetics of the model are compa
rable to those of the principal template of the model, P450BMP, as determin
ed from its crystallographic coordinates. The protein structure analysis pr
ograms PROCHECK, WHATIF, and SurVol indicate that the predicted P450c17 str
ucture is reasonable. The hydrophobic active site accommodates both Delta(4
) and Delta(5) steroid substrates in a catalytically favorable orientation.
The predicted contributions of positively charged residues to the redox-pa
rtner binding site were confirmed by site-directed mutagenesis. Molecular d
ynamic simulations with pregnenolone, 17-OH-pregnenolone, progesterone, and
Il-OH-progesterone docked into the substrate-binding pocket demonstrated t
hat regioselectivity of the hydroxylation reactions is determined both by p
roximity of hydrogens to the iron-ore complex and by the stability of the c
arbon radicals generated after hydrogen abstraction. The model explains the
activities of all known naturally occurring and synthetic human P450c17 mu
tants. The model predicted that mutation of lysine 89 would disrupt 17,20-l
yase activity to a greater extent than 17 alpha-hydroxylase activity; expre
ssion of a test mutant, K89N, in yeast confirmed this prediction. Hydrogen
peroxide did not support catalysis of the 17,20-lyase reaction, as would be
predicited by mechanisms involving a ferryl peroxide. Our present model an
d biochemical data suggest that both the hydroxylase and lyase activities p
roceed from a common steroid-binding geometry by an iron oxene mechanism. T
his model will facilitate studies of sex steroid synthesis and its disorder
s and the design of specific inhibitors useful in chemotherapy of sex stero
id-dependent cancers.