Physiological 3 beta-hydroxy-5-ene steroid substrates bind to 3 beta-hydroxysteroid dehydrogenase without the prior binding of cofactor.
Physiological 3 beta-hydroxy-5-ene steroid substrates bind to 3 beta-hydroxysteroid dehydrogenase without the prior binding of cofactor.
复制标题
生理性 3β-羟基-5-烯类固醇底物与 3β-羟基类固醇脱氢酶结合，无需事先结合辅因子。
DOI：
10.1016/0960-0760(96)00028-3
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Strickler,RC
中科院分区：
文献类型：
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作者：
Thomas,JL;Nash,WE;Strickler,RC
3- β-羟基-Δ5-steroid脱氢酶(3 - β- hsd)/类固醇Δ5−4异构酶催化人胎盘中3- β-羟基-5-烯类固醇（如孕烯醇酮）转化为3-氧-4-烯类固醇（孕酮）。利用NAD+/NADH和5α-还原类固醇5α-雄甾烷-3β、17β-二醇和5α-雄甾烷-17β-醇-3-酮在平衡状态下进行同位素交换，确定了这些3β-HSD底物[1]的辅因子一级结合。3β-羟基-5-烯甾体不能进行平衡交换，因为3β-HSD与5-烯底物不可逆。为了比较它们对辅助因子的结合需求，我们测试了3β-羟基-5-烯和3β-羟基-5α-还原类固醇在2α-溴乙酰孕酮（2α-BAP）存在或不存在辅助因子的情况下对纯化的人胎盘3β-HSD失活的保护作用。在没有辅助因子的孵育过程中，孕烯醇酮或脱氢表雄酮显著减缓（保护）了2α-BAP对3β-HSD的失活速率，2α-BAP是一种亲和烷基化物，可以特异性结合在3β-HSD底物位点。相比之下，5α-雄甾烷-3α-醇-17- 1、5α-雄甾烷-3β、17β-二醇、11α-乙酰氧基-5α-孕酮3,20-二酮仅在NADH （0.3 μM）或NAD+(10 μM)存在时才能保护3β-HSD免受2α-BAP的失活。在这些低浓度下，NADH和NAD+都不能减缓2α-BAP对3β-HSD的失活。此外，3-氧-5α-还原烷基化物11α-溴乙酰氧基-5α-孕酮3,20-二酮（11α-BA-5α-P）对3β-HSD没有特异性灭活作用。NAD+(10 μM)预孵育后，11α-BA-5α-P快速特异性灭活3β-HSD （t1 2= 3.7 min）。11α-溴乙氧基孕酮在NAD+存在或不存在的情况下，以相同的速率（t1 2= 5.0 min）灭活3β-HSD。这些亲和标记研究证实了3β-羟基-5α-还原类固醇的辅因子优先结合顺序，并最终表明更重要的生理3β-羟基-5-烯底物与3β-HSD结合不需要辅因子。
3β-Hydroxy-Δ5-steroid dehydrogenase (3β-HSD)/steroid Δ5−4-isomerase catalyses the conversion of 3β-hydroxy-5-ene steroids (e.g. pregnenolone) to 3-oxo-4-ene-steroids (progesterone) in human placenta. Isotope exchange at equilibrium using NAD+/NADH and the 5α-reduced steroids, 5α-androstane-3β, 17β-diol and 5α-androstan-17β-ol-3-one, determined a cofactor-first order of binding for these 3β-HSD substrates [1]. Exchange at equilibrium cannot be performed with 3β-hydroxy-5-ene steroids because 3β-HSD is not reversible with the 5-ene substrates. To compare their cofactor requirements for binding, 3β-hydroxy-5-ene and 3β-hydroxy-5α-reduced steroids were tested as protectors against the inactivation of purified human placental 3β-HSD by 2α-bromoacetoxyprogesterone (2α-BAP) in the presence or absence of cofactor. In incubations without cofactor, pregnenolone or dehydroepiandrosterone dramatically slowed (protected) the rate of 3β-HSD inactivation by 2α-BAP, an affinity alkylator that binds specifically at the 3β-HSD substrate site. In contrast, 5α-androstan-3α-ol-17-one, 5α-androstane-3β, 17β-diol, or 11α-acetoxy-5α-pregnan-3,20-dione protected 3β-HSD from inactivation by 2α-BAP only in the presence of NADH (0.3 μM) or NAD+(10 μM). At these low concentrations, neither NADH nor NAD+slowed the inactivation of 3β-HSD by 2α-BAP in the absence of protector-steroid. Further, the 3-oxo-5α-reduced alkylator, 11α-bromoacetoxy-5α-pregnan-3,20-dione (11α-BA-5α-P), did not inactivate 3β-HSD in a specific manner. After pre-incubation with NAD+(10 μM), 11α-BA-5α-P inactivated 3β-HSD rapidly and specifically (t1 2= 3.7 min ). 11α-Bromoacetoxyprogesterone inactivated 3β-HSD at the same rate (t1 2= 5.0 min ) in the presence or absence of NAD+. These affinity labelling studies confirm the cofactor-first binding order for 3β-hydroxy-5α-reduced steroids, and conclusively show that the more important, physiological 3β-hydroxy-5-ene substrates bind to 3β-HSD without a cofactor requirement.