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-[2H]-pregnenolone; 17-[2H]-, 16α-[2H]-, 21,21,21-[2H3]-, and 21-[2H]-progesterone; and 21,21,21-[2H3]-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-[2H]- and 17-[2H]-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-[2H3]-progesterone substrate. Competitive intermolecular KIEs paralleled the intramolecular KIE values, with DV values of 1.4–5.1 and DV/K values of 1.8–5.1 for these reactions. CYP17A1 and CYP21A2 mutation V359A both 16α-hydroxylate 16α-[2H]-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 non-linear and/or asymmetric, and C-H bond breakage is partially rate-limiting for all reactions.

Structures of Human Steroidogenic Cytochrome P450 17A1 with Substrates

“…1), which can compose 10 -30% of total hydroxylated metabolites depending on the system under study (17,46). For wild type CYP17A1 in the current purified enzyme system at saturating progesterone concentrations, 16␣-hydroxyprogesterone comprised 12% of the total progesterone hydroxylase activity, with 17␣-hydroxyprogesterone accounting for the remainder.…”

“…Human CYP17A1 has also been noted to produce very small amounts of 21-hydroxyprogesterone, also called 11-deoxycorticosterone, which is increased with the A105L mutation (46). Although the current purified system yielded no detectable 21-hydroxyprogesterone at the highest substrate concentrations used for determining steady-state kinetic parameters (70 M), at much higher progesterone concentrations (200 M), trace amounts of 21-hydroxyprogesterone were inconsistently detected for the A105L mutant but never for the wild type enzyme.…”

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

“…CYP17A1 and CYP21A2 Protein Expression and PurificationModified human (Gly 3 His 6 )-CYP17A1 was expressed in E. coli JM109 cells and purified as described (43,44). Modified human CYP21A2 was expressed in E. coli BL21(DE3) cells and purified as described (45).…”

“…Products were identified by retention times of external standards chromatographed at the beginning of the experiment. Data are analyzed as described previously (44). Percent product is calculated by product/(substrate ϩ product), which is multiplied by the starting concentration to determine amount.…”

Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5

“…Expression and Purification of Recombinant Proteins in Escherichia coli-Modified human CYP17A1 was expressed with GroEL/ES chaperones (pGro7 plasmid) in E. coli JM109 cells as described (17). Modified human POR was expressed in E. coli C41(DE3) cells (OverExpress, Lucigen, Middleton, WI) and purified according to the previously published procedure (18).…”