Division of Endocrinology & Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Biochemistry. 2012 Sep 11;51(36):7064-77. doi: 10.1021/bi300895w. Epub 2012 Aug 27.
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.
甾体羟化酶 CYP17A1（P450c17，17-羟化酶/17,20-裂合酶）和 CYP21A2（P450c21，21-羟化酶）在 2Å 半径内的一个或多个位点催化孕酮的羟化。我们用氘标记的底物探测了它们的氢原子提取机制和区域化学塑性：17-[(2)H]-孕烯醇酮；17-[(2)H]、16α-[(2)H]-、21、21、21-[(2)H(3)]-和 21-[(2)H]-孕酮；和 21、21、21-[(2)H(3)]-17-羟基孕酮。用重组人 P450-氧化还原酶和野生型人 CYP17A1 或突变 A105L（减少孕酮 16α-羟化）以及野生型人 CYP21A2 或突变 V359A（孕酮 16α-羟化的实质性增加）与野生型人 CYP21A2 或突变 V359A（孕酮 16α-羟化的实质性增加）进行产物分布和形成速率计算，计算出分子内和分子间的动力学同位素效应（KIEs）。CYP17A1 和突变 A105L 的分子内 KIE 分别为 H-17 的 4.1 和 3.8，H-16α 的 2.9 和 5.1。突变 A105L 21-羟化孕酮（产物的 5%），野生型 CYP17A1 也催化痕量的 21-羟化，这随着 16α-[(2)H]-和 17-[(2)H]-孕酮的增加而增加。CYP21A2 突变 V359A 和孕酮的分子内 KIE 分别为 H-21 和 H-16α 的 6.2 和 3.8。野生型 CYP21A2 也形成痕量的 16α-羟基孕酮，这随着 21、21、21-[(2)H(3)]-孕酮底物的增加而增加。竞争性的分子间 KIE 与分子内 KIE 值平行，这些反应的（D）V 值为 1.4-5.1，（D）V/K 值为 1.8-5.1。CYP17A1 和 CYP21A2 突变 V359A 均以 33-44%的氘保留 16α-[(2)H]-孕酮进行 16α-羟化，表明立体化学反转。我们得出结论，人 CYP17A1 具有孕酮 21-羟化酶活性，人 CYP21A2 具有孕酮 16α-羟化酶活性，这两种活性都通过氘代底物增强。这些羟化反应中 C-H 键断裂的过渡态要么明显非线性和/或不对称，并且 C-H 键断裂对于所有反应都是部分速率限制。
