Abstract

P450c17 and P450c21 are both type II mammalian cytochromes P450 that posses similar gene and protein structures and share 29% amino acid identity. Both P450s hydroxylate progesterone via carbon radical intermediates and an “oxygen rebound” mechanism. Progesterone hydroxylation by P450c17 and P450c21 is limited to positions on and near the D-ring of steroid substrates, specifically carbons 16, 17, and 21. Chemical principles dictate that reactions using carbon radical intermediates would favor hydroxylation at the 17 position, since tertiary carbon radicals are much more stable than secondary or primary radicals. Progesterone hydroxylation by P450c17 follows the expected pattern: 80% 17α-hydroxylation and 20% 16α-hydroxylation. Despite the similarities with P450c17, P450c21 hydroxylates only the disfavored 21 position of progesterone. The hydroxylase specificity differences between P450c17 and P450c21 derive from differences in active site structures, which manifests as different progesterone orientations. Since both P450s are membrane bound proteins that are difficult to crystallize, computer models of P450c17 and P450c21 have been built, using as templates the x-ray structures of P450-BMP and modified rabbit P450-2c5, respectively. After docking progesterone in the active sites of these models, molecular dynamics calculations demonstrate that progesterone binding parallel to the plane of the heme in P450c17 yields substrate trajectories that allow 16- and 17-hydroxylations. In the P450c21 model, however, progesterone must bind perpendicular to the heme to limit hydroxylation to the 21-carbon. The P450c21 models provide the opportunity to rationally design amino acid substitutions that increase active site volume, allowing progesterone to encounter additional trajectories that allow 16- and/or 17-hydroxylations. Our lab has engineered a P450c21 mutation (figure 4) with 16-hydroxylase activity by substituting I447A and V336A (P450c21mut). Hypothesis: P450c17 is a 17- and 16-hydroxylase because this enzyme can hydroxylate multiple sites on progesterone, and activities reflect thermodynamic preference. P450c21 is a 21-hydroxylase because this is the only position accessible to the heme oxene. Approach: Introduce deuterium atoms at the sites of hydroxylation to disfavor those sites by kinetic isotope effects. In this poster, we present results with [21, 21, 21, 17-D4]-progesterone as substrate for WT and mutant P450c21 and with 17-D-progesterone as substrate for P450c17.