Minor Activities and Transition State Properties of the Human Steroid Hydroxylases Cytochromes P450c17 and P450c21, from Reactions Observed with Deuterium-Labeled Substrates

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 angstrom radius. We probed their hydrogen atom abstraction mechanisms and regiochemical plasticity with deuterium-labeled substrates: 17-[H-2]-pregnenolone; 16 alpha-[H-2]-, 21,21,21-[H-2(3)]-, and 21-[H-2-]progesterone; and 21,21,21-[H-2(3)]-17-hydroxyprogesterone. Product distribution and formation rates with recombinant human P450-oxidoreductase and wild-type human CYP17A1 or mutation A105L (reduced progesterone 16 alpha-hydroxylation) and wild-type human CYP21A2 or mutation V359A (substantial progesterone 16 alpha-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 alpha. Mutation A105L 21-hydroxylates progesterone (5% of products), and wild-type CYP17A1 also catalyzes a trace of 21-hydroxylation, which increases with 16 alpha-[H-2]- and 17-[H-2]-progesterone. The intramolecular KIEs with CYP21A2 mutation V359A and progesterone were 6.2 and 3.8 at H-21 and H-16 alpha, respectively. Wild-type CYP21A2 also forms a trace of 16 alpha-hydroxyprogesterone, which increased with 21,21,21-[H-2(3)]-progesterone substrate. Competitive intermolecular KIEs paralleled the intramolecular KIE values, with V-D values of 1.4-5.1 and V-D/K values of 1.8-5.1 for these reactions. CYP17A1 and CYP21A2 mutation V359A both 16 alpha-hydroxylate 16 alpha-[H-2]-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 alpha-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.