title: Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates.
abstract: The steroid hydroxylases CYP17A1 (P450c17, 17-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) catalyze progesterone hydroxylation at one or more sites within a 2 Å radius. We probed their hydrogen atom abstraction mechanisms and regiochemical plasticity with deuterium-labeled substrates: 17-[(2)H]-pregnenolone; 17-[(2)H]-, 16α-[(2)H]-, 21,21,21-[(2)H(3)]-, and 21-[(2)H]-progesterone; and 21,21,21-[(2)H(3)]-17-hydroxyprogesterone. Product distribution and formation rates with recombinant human P450-oxidoreductase and wild-type human CYP17A1 or mutation A105L (reduced progesterone 16α-hydroxylation) and wild-type human CYP21A2 or mutation V359A (substantial progesterone 16α-hydroxylation) were used to calculate intramolecular and intermolecular kinetic isotope effects (KIEs). The intramolecular KIEs for CYP17A1 and mutation A105L were 4.1 and 3.8, respectively, at H-17 and 2.9 and 5.1, respectively, at H-16α. Mutation A105L 21-hydroxylates progesterone (5% of products), and wild-type CYP17A1 also catalyzes a trace of 21-hydroxylation, which increases with 16α-[(2)H]- and 17-[(2)H]-progesterone. The intramolecular KIEs with CYP21A2 mutation V359A and progesterone were 6.2 and 3.8 at H-21 and H-16α, respectively. Wild-type CYP21A2 also forms a trace of 16α-hydroxyprogesterone, which increased with 21,21,21-[(2)H(3)]-progesterone substrate. Competitive intermolecular KIEs paralleled the intramolecular KIE values, with (D)V values of 1.4-5.1 and (D)V/K values of 1.8-5.1 for these reactions. CYP17A1 and CYP21A2 mutation V359A both 16α-hydroxylate 16α-[(2)H]-progesterone with 33-44% deuterium retention, indicating stereochemical inversion. We conclude that human CYP17A1 has progesterone 21-hydroxylase activity and human CYP21A2 has progesterone 16α-hydroxylase activity, both of which are enhanced with deuterated substrates. The transition states for C-H bond cleavage in these hydroxylation reactions are either significantly nonlinear and/or asymmetric, and C-H bond breakage is partially rate-limiting for all reactions.
authors.affiliation: Division of Endocrinology & Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Francis K
authors.initials: FK
authors.lastname: Yoshimoto
authors.forename: Yishan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Hwei-Ming
authors.initials: HM
authors.lastname: Peng
authors.forename: David
authors.initials: D
authors.lastname: Stidd
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Yoshimoto
authors.forename: Kamalesh K
authors.initials: KK
authors.lastname: Sharma
authors.forename: Susan
authors.initials: S
authors.lastname: Matthew
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Auchus
mesh_terms: D003903:Deuterium / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000378:metabolism*; D013255:Steroid 21-Hydroxylase / Q000737:chemistry* / Q000378:metabolism*; D013256:Steroids / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D013256:Steroids; D003903:Deuterium; D013255:Steroid 21-Hydroxylase; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase