STEREOSPECIFICITY OF HYDROGEN TRANSFER BETWEEN PROGESTERONE AND COFACTOR BY HUMAN PLACENTAL ESTRADIOL-17-BETA DEHYDROGENASE

We have previously shown that human placental estradiol-17-beta dehydrogenase (EC 1.1.1.62; 17-beta-EDH) catalyzes the conversion of estradiol-17-beta 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-H-3]NADPH, was generated with homogeneous 17-beta-EDH from term human placenta, utilizing [17-alpha-H-3]estradiol-17-beta and NADP+. The resulting [4B-H-3]NADPH was then purified by ion exchange chromatography and was separately incubated (24.4-mu-M) with a large molar excess of progesterone (150-mu-M) 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 20-alpha-hydroxy-4-pregnen-3-one (20-alpha-OHP) and NADP+ were obtained. Radioactivity was stoichiometrically transferred from [4B-H-3]NADPH to the steriod product ([H-3]20-alpha-OHP). These results further substantiate a single active site for both 17-beta- and 20-alpha-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 20-alpha-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 sterospecificity.