title: Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase: STRUCTURE OF THE ENZYME·PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING C-H BOND CLEAVAGE.
abstract: Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼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 17α-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 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, 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 (kon 2.4 × 10(7) M(-1) s(-1)) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 10(7) M(-1) s(-1)) with this substrate, suggesting 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.
authors: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and.
authors: Pradeep S
authors: PS
authors: Pallan
authors: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and.
authors: Chunxue
authors: C
authors: Wang
authors: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and.
authors: Li
authors: L
authors: Lei
authors: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and.
authors: Francis K
authors: FK
authors: Yoshimoto
authors: the Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors: Richard J
authors: RJ
authors: Auchus
authors: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and.
authors: Michael R
authors: MR
authors: Waterman
authors: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and f.guengerich@vanderbilt.edu.
authors: F Peter
authors: FP
authors: Guengerich
authors: From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and martin.egli@vanderbilt.edu.
authors: Martin
authors: M
authors: Egli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000737:chemistry; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011374:Progesterone / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013255:Steroid 21-Hydroxylase / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011374:Progesterone; D003903:Deuterium; C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase
keywords: cytochrome P450; enzyme kinetics; isotope effect; steroidogenesis; x-ray crystallography