Stereospecificity of hydrogen transfer between progesterone and cofactor by human placental estradiol-17β dehydrogenase

Abstract

We have previously shown that human placental estradiol-17β dehydrogenase (EC 1.1.1.62; 17β-EDH) catalyzes the conversion of estradiol-17β to estrone and stereospecifically reduces NAD + to [4-pro-S]NADH, ([4-B]NADH). Subsequently, this enzyme was found to reduce the ketone function at C-20 of progesterone, and evidence indicates that both activities reside at the same active site. This study was done to further elucidate spatial arrangements of cofactor and the 21-carbon substrate as they bind at the active site. The cofactor, [4B- 3H]NADPH, was generated with homogeneous 17β-EDH from term human placenta, utilizing [17α- 3H]estradiol-17β and NADP +. The resulting [4B- 3H]NADPH was then purified by ion exchange chromatography and was separately incubated (24.4 μM) with a large molar excess of progesterone (150 μM) as substrate in the presence of the enzyme. Following incubation, the steroid reactants and products were extracted, separated by high-performance liquid chromatography and quantitated as to mass and tritium content. Oxidized and reduced cofactor were separated by ion-exchange chromatography and similarly quantitated. In all incubations, equimolar amounts of 20α-hydroxy-4-pregnen-3-one (20α-OHP) and NADP + were obtained. Radioactivity was stoichiometrically transferred from [4B- 3H]NADPH to the steroid product ([ 3H]20α-OHP). These results further substantiate a single active site for both 17β- and 20α-dehydrogenation enzyme activities. In addition, the enzyme is B-side specific, catalyzing the transfer of the 4B-hydrogen from the dihydronicotinamide moiety of the cofactor, for both C-18 and C-21 steroid substrates. Since the 20α-dehydrogenation by other enzyme sources has always been demonstrated to be an A-side specific reaction, this observation represents an important exception to the Alworth-Bentley rules of enzyme stereospecificity.