Kinetics of allopregnanolone formation catalyzed by human 3 alpha-hydroxysteroid dehydrogenase type III (AKR1C2).
Kinetics of allopregnanolone formation catalyzed by human 3 alpha-hydroxysteroid dehydrogenase type III (AKR1C2).
复制标题
由人 3 α-羟基类固醇脱氢酶 III 型 (AKR1C2) 催化的四氢孕酮形成动力学。
作者：
J. Trauger;A. Jiang;B. Stearns;P. Lograsso
Allopregnanolone 是一种神经类固醇，通过增强 GABA(A) 受体发挥抗焦虑和抗惊厥活性。 5α-二氢孕酮 (5α-DHP) 的还原是四氢孕酮生物合成的最后一步，由 3α-羟基类固醇脱氢酶 (3α-HSD) 催化。虽然四氢孕酮的作用机制以及体内四氢孕酮浓度的生理学和药理学调节已被广泛研究，但对人 3α-HSD 催化四氢孕酮形成的动力学特征却很少。我们在此报告通过使用稳态动力学研究确定人 3α-HSD III 型催化 5α-DHP 还原的动力学机制，并评估氟西汀和各种其他小分子激活 3α-HSD III 型催化四氢孕酮形成的能力。酶催化的 5α-DHP 还原产生两种产物，四氢孕酮和 5α,20α-四氢孕酮（通过使用放射性薄层色谱测定进行测量），而 5β-DHP 还原产生神经类固醇孕酮，这是唯一的产物。 5β-DHP还原的催化效率比5α-DHP还原高10倍。双底物动力学分析和 5α-DHP 还原和四氢孕酮氧化的死端抑制研究表明，III 型 3α-HSD 采用三元复合（顺序）动力学机制，烟酰胺腺嘌呤二核苷酸辅因子在类固醇底物之前结合，在类固醇产物之后离开。由于之前的报道表明氟西汀和某些其他小分子通过激活 III 型 3α-HSD 增加体内异孕酮浓度，因此我们研究了这些小分子是否能够激活人 III 型 3α-HSD。我们的结果表明，在浓度高达 50 µM 时，氟西汀、帕罗西汀、舍曲林、去甲氟西汀、卡马西平、氯氮平、氟比洛芬和磺溴酞不会激活该酶。这些结果表征了 III 型 3α-HSD 在四氢孕酮形成中的作用，并表明氟西汀激活该酶可能不是氟西汀增加四氢孕酮浓度的机制。
Allopregnanolone is a neurosteroid which exhibits anxiolytic and anticonvulsant activities through potentiation of the GABA(A) receptor. The reduction of 5alpha-dihydroprogesterone (5alpha-DHP), the last step in allopregnanolone biosynthesis, is catalyzed by 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs). While the mechanism of action of allopregnanolone and the physiological and pharmacological modulation of allopregnanolone concentrations in vivo have been extensively studied, there has been little characterization of the kinetics of human 3alpha-HSD catalyzed allopregnanolone formation. We report here determination of the kinetic mechanism for 5alpha-DHP reduction catalyzed by human 3alpha-HSD type III by using steady-state kinetics studies and assessment of the ability of fluoxetine and various other small molecules to activate 3alpha-HSD type III catalyzed allopregnanolone formation. Enzyme-catalyzed 5alpha-DHP reduction yielded two products, allopregnanolone and 5alpha,20alpha-tetrahydroprogesterone, as measured by using a radiometric thin-layer chromatography assay, while 5beta-DHP reduction yielded the neurosteroid pregnanolone as the only product. 5Beta-DHP reduction proceeded with a catalytic efficiency 10 times higher than that of 5alpha-DHP reduction. Two-substrate kinetic analysis and dead-end inhibition studies for 5alpha-DHP reduction and allopregnanolone oxidation indicated that 3alpha-HSD type III utilized a ternary complex (sequential) kinetic mechanism, with nicotinamide adenine dinucleotide cofactor binding before steroid substrate and leaving after steroid product. Since previous reports suggested that fluoxetine and certain other small molecules increased allopregnanolone concentrations in vivo by activating 3alpha-HSD type III, we investigated whether these small molecules were able to activate human 3alpha-HSD type III. Our results showed that, at concentrations up to 50 microM, fluoxetine, paroxetine, sertraline, norfluoxetine, carbamazepine, clozapine, flurbiprofen, and sulfobromophthalein did not activate the enzyme. These results characterize the role of 3alpha-HSD type III in allopregnanolone formation and suggest that activation of this enzyme by fluoxetine is likely not the mechanism by which fluoxetine increases allopregnanolone concentrations.