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-3H]NADPH, was generated with homogeneous 17 beta-EDH from term human placenta, utilizing [17 alpha-3H]estradiol-17 beta and NADP+. The resulting [4B-3H]NADPH was then purified by ion exchange chromatography and was separately incubated (24.4 microM) with a large molar excess of progesterone (150 microM) 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-3H]NADPH to the steroid product [( 3H]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 stereospecificity.
Pineda
J A
JA
Department of Obstetrics and Gynecology, Washington University School of Medicine, St Louis 63110.
Murdock
G L
GL
Watson
R J
RJ
Warren
J C
JC
D001665:Binding Sites; D001692:Biological Transport; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004958:Estradiol / Q000378:metabolism; D004960:Estradiol Dehydrogenases / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009536:Niacinamide / Q000031:analogs & derivatives / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011374:Progesterone / Q000378:metabolism*; D013379:Substrate Specificity
C015145:dihydronicotinamide; D009536:Niacinamide; D011374:Progesterone; D004958:Estradiol; D009249:NADP; D006859:Hydrogen; D004960:Estradiol Dehydrogenases; D005969:Glutamate Dehydrogenase