Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase STRUCTURE OF THE ENZYME.PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING C-H BOND CLEAVAGE

Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in similar to 95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17 alpha-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17 alpha-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both k(cat) and k(cat)/K-m, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (k(on) 2.4 x 10(7) M-1 s(-1)) is only similar to 2-fold greater than the catalytic efficiency (k(cat)/K-m = 1.3 x 10(7) M-1 s(-1)) with this substrate, sug-gesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.