Trauger John W, Jiang Alice, Stearns Brian A, LoGrasso Philip V
Department of Molecular Neuroscience, Merck Research Laboratories, 3535 General Atomics Court, San Diego, California 92121, USA.
Biochemistry. 2002 Nov 12;41(45):13451-9. doi: 10.1021/bi026109w.
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.
别孕烯醇酮是一种神经甾体，通过增强γ-氨基丁酸A（GABA(A)）受体而表现出抗焦虑和抗惊厥活性。别孕烯醇酮生物合成的最后一步，即5α-二氢孕酮（5α-DHP）的还原，由3α-羟基类固醇脱氢酶（3α-HSDs）催化。虽然对别孕烯醇酮的作用机制以及体内别孕烯醇酮浓度的生理和药理调节已进行了广泛研究，但对人3α-HSD催化别孕烯醇酮形成的动力学特性了解甚少。我们在此报告，通过稳态动力学研究以及评估氟西汀和其他各种小分子激活Ⅲ型人3α-HSD催化别孕烯醇酮形成的能力，确定Ⅲ型人3α-HSD催化5α-DHP还原的动力学机制。通过放射性薄层色谱分析测定，酶催化的5α-DHP还原产生两种产物，别孕烯醇酮和5α,20α-四氢孕酮，而5β-DHP还原仅产生神经甾体孕烷醇酮作为唯一产物。5β-DHP还原的催化效率比5α-DHP还原高10倍。对5α-DHP还原和别孕烯醇酮氧化的双底物动力学分析及终产物抑制研究表明，Ⅲ型3α-HSD采用三元复合物（顺序）动力学机制，烟酰胺腺嘌呤二核苷酸辅因子在甾体底物之前结合，在甾体产物之后离开。由于先前的报告表明氟西汀和某些其他小分子通过激活Ⅲ型3α-HSD增加体内别孕烯醇酮浓度，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、氟比洛芬和磺溴酞钠均未激活该酶。这些结果阐明了Ⅲ型3α-HSD在别孕烯醇酮形成中的作用，并表明氟西汀激活该酶可能不是其增加别孕烯醇酮浓度的机制。
