Kinetics of allopregnanolone formation catalyzed by human 3 alpha-hydroxysteroid dehydrogenase type III (AKR1C2).
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.
Trauger
John W
JW
Department of Molecular Neuroscience, Merck Research Laboratories, 3535 General Atomics Court, San Diego, California 92121, USA.
Jiang
Alice
A
Stearns
Brian A
BA
LoGrasso
Philip V
PV
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D043582:5-alpha-Dihydroprogesterone; D002220:Carbamazepine / Q000737:chemistry; D002384:Catalysis; D003024:Clozapine / Q000737:chemistry; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D005473:Fluoxetine / Q000031:analogs & derivatives* / Q000737:chemistry; D005480:Flurbiprofen / Q000737:chemistry; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D011277:Pregnanediones / Q000737:chemistry; D011280:Pregnanolone / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification; D017367:Selective Serotonin Reuptake Inhibitors / Q000737:chemistry; D013379:Substrate Specificity; D014580:Ursodeoxycholic Acid / Q000737:chemistry
D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D043582:5-alpha-Dihydroprogesterone; D002220:Carbamazepine; D003024:Clozapine; D004791:Enzyme Inhibitors; D005473:Fluoxetine; D005480:Flurbiprofen; D006913:Hydroxysteroid Dehydrogenases; D011277:Pregnanediones; D011280:Pregnanolone; D011993:Recombinant Fusion Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D014580:Ursodeoxycholic Acid; C435496:AKR1C2 protein, human; C036139:norfluoxetine