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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]-, 16?-[(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 16?-hydroxylation) and wild-type human CYP21A2 or mutation V359A (substantial progesterone 16?-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-16?. Mutation A105L 21-hydroxylates progesterone (5% of products), and wild-type CYP17A1 also catalyzes a trace of 21-hydroxylation, which increases with 16?-[(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-16?, respectively. Wild-type CYP21A2 also forms a trace of 16?-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 16?-hydroxylate 16?-[(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 16?-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.

SUBMITTER: Yoshimoto FK

PROVIDER: S-EPMC3471366 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates.

Yoshimoto Francis K FK Zhou Yishan Y Peng Hwei-Ming HM Stidd David D Yoshimoto Jennifer A JA Sharma Kamalesh K KK Matthew Susan S Auchus Richard J RJ

Biochemistry 20120827 36

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 recom ...[more]

PMID: 22873692

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Dataset Information

Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates.

Publications

Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates.

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