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P450c17 (17alpha-hydroxylase/17,20-lyase) catalyzes steroid 17alpha-hydroxylase
and 17,20-lyase activities in the biosynthesis of androgens and estrogens. These
two activities are differentially regulated in a tissue-specific and
developmentally programmed manner. To visualize the active site topology of
human P450c17 and to study the structural basis of its substrate specificity and
catalytic selectivity, we constructed a second-generation computer-graphic model
of human P450c17. The energetics of the model are comparable to those of the
principal template of the model, P450BMP, as determined from its
crystallographic coordinates. The protein structure analysis programs PROCHECK,
WHATIF, and SurVol indicate that the predicted P450c17 structure is reasonable.
The hydrophobic active site accommodates both delta4 and delta5 steroid
substrates in a catalytically favorable orientation. The predicted contributions
of positively charged residues to the redox-partner binding site were confirmed
by site-directed mutagenesis. Molecular dynamic simulations with pregnenolone,
17-OH-pregnenolone, progesterone, and 17-OH-progesterone docked into the
substrate-binding pocket demonstrated that regioselectivity of the hydroxylation
reactions is determined both by proximity of hydrogens to the iron-oxo complex
and by the stability of the carbon radicals generated after hydrogen
abstraction. The model explains the activities of all known naturally occurring
and synthetic human P450c17 mutants. The model predicted that mutation of lysine
89 would disrupt 17,20-lyase activity to a greater extent than
17alpha-hydroxylase activity; expression 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 predicted by mechanisms involving a ferryl
peroxide. Our present model and biochemical data suggest that both the
hydroxylase and lyase activities proceed from a common steroid-binding geometry
by an iron oxene mechanism. This model will facilitate studies of sex steroid
synthesis and its disorders and the design of specific inhibitors useful in
chemotherapy of sex steroid-dependent cancers.
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