Mechanistic Studies of Two Steroidogenic Cytochromes P450, CYP17A1 and CYP21A2




Yoshimoto, Francis Kiichi Iida

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Human CYP17A1 (P450c17 or 17alpha-hydroxylase/17,20-lyase) is a member of the P450 superfamily of proteins. This enzyme is responsible for hydroxylating the 17-position of pregnenolone or progesterone and cleaving the 17,20-carbon,carbon-bond of its hydroxylated products, which leads to the formation of androgens. The latter activity makes the inhibition of this enzyme a target for the treatment of prostate cancer. Human CYP21A2 (P450c21 or 21-hydroxylase) hydroxylates the 21-position of progesterone and 17-hydroxyprogesterone – deficiency in this enzyme leads to congenital adrenal hyperplasia. Intermolecular and intramolecular kinetic isotope effects were determined for both enzymes, which required the synthesis of regioselectively deuterated steroids: 17alpha-[2H]-progesterone, 17alpha-[2H]-pregnenolone, 21,21,21-[2H3]-progesterone, 21-[2H]-progesterone, 16alpha-[2H]-progesterone and 21,21,21-[2H3]-17-hydroxyprogesterone. Based on the calculated isotope effects the C-H abstraction step was determined to be partially rate-limiting in the overall hydroxylation process. Moreover, novel 21-hydroxylase activity and 16-hydroxylase activity on progesterone was observed for CYP17A1 and CYP21A2, respectively. CYP17A1 catalyzed hydroxylation of progesterone on the 21:17:16 positions in a 3.4:76.3:20.3 ratio, and this ratio changed to 5.5:87.4:7.1 when 16alpha-[2H]-progesterone was used as the substrate. Meanwhile, CYP21A2 hydroxylated the 21:16 positions in a 99.6:0.4 ratio and this ratio changed to 94.3:5.7 when 21,21,21-[2H3]-progesterone was used as the substrate. Kinetic isotope effects with 17alpha-[3H]-pregnenolone were calculated for CYP17A1. Olefinated progesterone analogs were synthesized to probe possible epoxidase activity of these steroidogenic enzymes. CYP17A1 hydroxylated the 21-position and epoxidized the 16alpha,17alpha-position of 16,17-dehydroprogesterone while CYP21A2 was found to only hydroxylate the 21-position of the same substrate. Both CYP17A1 and CYP21A2 reduced the 21,22-alkene of 21-homo-21,22-dehydroprogesterone and further hydroxylated the metabolite. Other potential substrates such as cyclopropyl and halogenated progesterone analogs were also synthesized to study the enzymatic reactivity towards these substrates. CYP17A1 was found to metabolize 17beta-cyclopropylmethylandrostenone and CYP21A2 was found to metabolize 17-fluoroprogesterone. Both enzymes were found to metabolize 20-desoxoprogesterone – each enzyme yielded a different product with this substrate. Exploring the mechanistic behavior of these enzymes towards various steroid analogs enhances our understanding of their reactivities and structural properties. This research ultimately provides insights on more detailed roles of these enzymes in human disease and may help us to design better inhibitors.

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