Kinetics of Allopregnanolone Formation Catalyzed by Human 3-Hydroxysteroid Dehydrogenase Type III (AKR1C2)
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- 作者：John W. Trauger ; Alice Jiang ; Brian A. Stearns ; and Philip V. LoGrasso
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：November 12, 2002
- 年：2002
- 卷：41
- 期：45
- 页码：13451 - 13459
- 全文大小：153K
- 年卷期：v.41,no.45(November 12, 2002)
- ISSN：1520-4995
文摘
Allopregnanolone is a neurosteroid which exhibits anxiolytic and anticonvulsant activitiesthrough potentiation of the GABAA receptor. The reduction of 5
-dihydroprogesterone (5-DHP), thelast step in allopregnanolone biosynthesis, is catalyzed by 3-hydroxysteroid dehydrogenases (3-HSDs).While the mechanism of action of allopregnanolone and the physiological and pharmacological modulationof allopregnanolone concentrations in vivo have been extensively studied, there has been littlecharacterization of the kinetics of human 3-HSD catalyzed allopregnanolone formation. We report heredetermination of the kinetic mechanism for 5-DHP reduction catalyzed by human 3-HSD type III byusing steady-state kinetics studies and assessment of the ability of fluoxetine and various other smallmolecules to activate 3-HSD type III catalyzed allopregnanolone formation. Enzyme-catalyzed 5-DHP reduction yielded two products, allopregnanolone and 5,20-tetrahydroprogesterone, as measuredby using a radiometric thin-layer chromatography assay, while 5
-DHP reduction yielded the neurosteroidpregnanolone as the only product. 5-DHP reduction proceeded with a catalytic efficiency 10 times higherthan that of 5-DHP reduction. Two-substrate kinetic analysis and dead-end inhibition studies for 5-DHP reduction and allopregnanolone oxidation indicated that 3-HSD type III utilized a ternary complex(sequential) kinetic mechanism, with nicotinamide adenine dinucleotide cofactor binding before steroidsubstrate and leaving after steroid product. Since previous reports suggested that fluoxetine and certainother small molecules increased allopregnanolone concentrations in vivo by activating 3-HSD type III,we investigated whether these small molecules were able to activate human 3-HSD type III. Our resultsshowed that, at concentrations up to 50 
M, fluoxetine, paroxetine, sertraline, norfluoxetine, carbamazepine,clozapine, flurbiprofen, and sulfobromophthalein did not activate the enzyme. These results characterizethe role of 3-HSD type III in allopregnanolone formation and suggest that activation of this enzyme byfluoxetine is likely not the mechanism by which fluoxetine increases allopregnanolone concentrations.
-dihydroprogesterone (5-DHP), thelast step in allopregnanolone biosynthesis, is catalyzed by 3-hydroxysteroid dehydrogenases (3-HSDs).While the mechanism of action of allopregnanolone and the physiological and pharmacological modulationof allopregnanolone concentrations in vivo have been extensively studied, there has been littlecharacterization of the kinetics of human 3-HSD catalyzed allopregnanolone formation. We report heredetermination of the kinetic mechanism for 5-DHP reduction catalyzed by human 3-HSD type III byusing steady-state kinetics studies and assessment of the ability of fluoxetine and various other smallmolecules to activate 3-HSD type III catalyzed allopregnanolone formation. Enzyme-catalyzed 5-DHP reduction yielded two products, allopregnanolone and 5,20-tetrahydroprogesterone, as measuredby using a radiometric thin-layer chromatography assay, while 5-DHP reduction yielded the neurosteroidpregnanolone as the only product. 5-DHP reduction proceeded with a catalytic efficiency 10 times higherthan that of 5-DHP reduction. Two-substrate kinetic analysis and dead-end inhibition studies for 5-DHP reduction and allopregnanolone oxidation indicated that 3-HSD type III utilized a ternary complex(sequential) kinetic mechanism, with nicotinamide adenine dinucleotide cofactor binding before steroidsubstrate and leaving after steroid product. Since previous reports suggested that fluoxetine and certainother small molecules increased allopregnanolone concentrations in vivo by activating 3-HSD type III,we investigated whether these small molecules were able to activate human 3-HSD type III. Our resultsshowed that, at concentrations up to 50 M, fluoxetine, paroxetine, sertraline, norfluoxetine, carbamazepine,clozapine, flurbiprofen, and sulfobromophthalein did not activate the enzyme. These results characterizethe role of 3-HSD type III in allopregnanolone formation and suggest that activation of this enzyme byfluoxetine is likely not the mechanism by which fluoxetine increases allopregnanolone concentrations.