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-Hydroxylase/ 17,20-Lyase): Insights into Reaction Mechanisms and Effects of Mutations
Departments of Pediatrics (R.J.A., W.L.M.) and Internal Medicine (R.J.A.) and The Metabolic Research Unit (W.L.M.) University of California San Francisco, California 94143-0978
P450c17 (17
-hydroxylase/17,20-lyase) catalyzes
steroid 17-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 
4
and 5 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
17-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 predicited 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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W. L. Miller and R. J. Auchus Role of Cytochrome b5 in the 17,20-Lyase Activity of P450c17 J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1346 - 1346. [Full Text]
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G. I. Lepesheva, L. M. Podust, A. Bellamine, and M. R. Waterman Folding Requirements Are Different between Sterol 14alpha -Demethylase (CYP51) from Mycobacterium tuberculosis and Human or Fungal Orthologs J. Biol. Chem., July 20, 2001; 276(30): 28413 - 28420. [Abstract] [Full Text] [PDF]
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