Stereospecificity of hydrogen transfer between progesterone and cofactor by human placental estradiol-17 beta dehydrogenase.
Stereospecificity of hydrogen transfer between progesterone and cofactor by human placental estradiol-17 beta dehydrogenase.
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人胎盘雌二醇-17β脱氢酶在孕酮和辅因子之间氢转移的立体特异性。
DOI：
10.1016/0960-0760(90)90373-s
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Warren,JC
中科院分区：
文献类型：
--
作者：
Pineda,JA;Murdock,GL;Watson,RJ;Warren,JC
我们先前已经证明，人胎盘雌二醇-17β脱氢酶(EC 1.1.1.62；17β-EDH)催化雌二醇-17β转化为雌酮，并立体特异性地将NAD+还原为[4-Pro-S]NADH，([4-B]NADH)。随后，这种酶被发现降低了孕酮的C-20位上的酮功能，并且有证据表明，这两种活性都位于同一活性部位。这项研究是为了进一步阐明辅因子和21碳底物在活性部位结合时的空间排列。辅因子[4B-~3H]NADPH是从足月人胎盘中用均一的17β-EDH产生的，利用[17α-~3H]雌二醇-17β和NADP+。经离子交换层析纯化后，以大摩尔过量孕酮(150μM)为底物，单独孵育(2 4.4μM)。孵育后，提取类固醇反应物和产物，用高效液相色谱分离，并对质量和氚含量进行定量。用离子交换层析分离氧化和还原辅因子，并进行类似的定量。在所有培养条件下，均可获得等摩尔量的20α-羟基-4-孕烯-3-酮(20α-OHP)和NADP+。放射性从[4B-~3H]α转移到类固醇产物([~3H]20 NADPH-OHP)。这些结果进一步证实了17个β-脱氢酶和20个α-脱氢酶的单一活性部位。此外，对于C-18和C-21类固醇底物，该酶是B侧专一性的，催化从辅因子的二氢烟酰胺部分转移4B-氢。由于其他酶来源的20α脱氢一直被证明是A侧特异反应，这一观察到的结果是对阿尔沃斯-本特利酶立体专一性规则的一个重要例外。
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.