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.

Figure 1

Major steroid hydroxylase activities of human CYP17A1 and CYP21A2 with principal substrates. The 17,20-lyase reactions catalyzed by CYP17A1 are omitted for simplicity.

Figure 2

Intramolecular KIEs and metabolic switching with wild-type human CYP17A1 (A) and mutation A105L (B). HPLC tracings show products obtained by incubation with microsomal enzyme system with (top to bottom) 16α-[²H]-, 17-[²H]-, 21,21,21-[²H₃]-, and natural abundance progesterone (P4) substrates. Products were identified by retention times of standards chromatographed before and after samples: 1, 16α-hydroxyprogesterone; 2, 21-hydroxyprogesterone (DOC); 3, 17-hydroxyprogesterone; 4, progesterone. Ordinate scales are 0.15–0.25 and 0.08–0.25 AU full scale for Panels A and B, respectively.

Figure 3

Intermolecular KIE data with wild-type human CYP17A1 (A) and mutation A105L (B). Chromatograms show radioactivity (top, 400–2500 CPM full scale) and absorbance at 254 nm (bottom, 0.08–1.8 AU full scale) derived from co-incubations with deuterium-labeled steroid and tracer tritium-labeled steroid as indicated. Products were identified by retention times of standards chromatographed before and after samples, same key as Figure 2: 1, 16α-hydroxyprogesterone; 2, DOC; 3, 17-hydroxyprogesterone; 4, progesterone (P4). Experiments with pregnenolone were followed by cholesterol oxidase treatment prior to analysis.

Figure 4

Intramolecular KIEs and metabolic switching with wild-type human CYP21A2 (A) and mutation V359A (B). HPLC tracings show products obtained by incubation with microsomal enzyme system with (top to bottom) 16α-[²H]-, 21,21,21-[²H₃]-, and natural abundance progesterone (P4) substrates. Products were identified by retention times of standards chromatographed before and after samples same key as Figure 2: 1, 16α-hydroxyprogesterone; 2, DOC; 4, progesterone (P4). Ordinate scales are 0.02–0.08 AU full scale. Peaks near 16α-hydroxyprogesterone (1) derived from incubations with 16α-[²H]-progesterone in A are trace contaminants in substrate with different retention times than 1.

Figure 5

Intermolecular KIE data with wild-type human CYP21A2 (A) and mutation V359A (B). Chromatograms show radioactivity (top, 800–2000 CPM full scale) and absorbance at 254 nm (bottom, 0.2–0.6 AU full scale; inset in Panel A is 0.03 AU full scale) derived from co-incubations with deuterium-labeled steroid and tracer tritium-labeled steroid as indicated. Products were identified by retention times of standards chromatographed before and after samples, same key as Figure 2 [1, 16α-hydroxyprogesterone; 2, DOC; 3, 17-hydroxyprogesterone; 4, progesterone (P4)] plus 5, 11-deoxycortisol; 6, 16α,17α-dihydroxyprogesterone (pregn-4-ene-16α,17α-diol-3,20-dione). A trace of 6 appears to be produced by mutation V359A (lower right of B).

Scheme 1

Synthesis of 16α-[²H]-progesterone (5).

Scheme 2

Synthesis of 17-[²H]-pregnenolone (7), 17-[²H]-progesterone (9), and 21,21,21-[²H₃]-progesterone (11).

Scheme 3

Synthesis of 21,21,21-[²H₃]-17-hydroxyprogesterone (14).