Analysis of coenzyme binding by human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase using 5'-[p-(fluorosulfonyl)benzoyl]adenosine, an affinity labeling cofactor analog.
Analysis of coenzyme binding by human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase using 5'-[p-(fluorosulfonyl)benzoyl]adenosine, an affinity labeling cofactor analog.
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使用 5-[p-(氟磺酰基)苯甲酰基]腺苷（一种亲和标记辅因子类似物）分析人胎盘 3β-羟基-5-烯-类固醇脱氢酶和类固醇 5----4-烯-异构酶的辅酶结合。
DOI：
10.1016/0960-0760(91)90240-6
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发表时间：
1991
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;Myers,RP;Strickler,RC
3β-羟基-5-烯-类固醇脱氢酶和类固醇5→4-烯-异构酶作为单一、同质的蛋白质从人胎盘微粒体中共纯化。与2α-溴乙酰氧基孕酮的亲和烷基化表明脱氢酶和异构酶底物类固醇结合在同一蛋白质的不同位点。然而，辅酶NADH完全消除了由胰蛋白酶类似物引起的两种酶活性的烷基化[托马斯J.L.，迈尔斯河P.，罗西克湖O.和Strickler R. C.的方法，36（1990）117-123]。与细菌3-酮-5-烯-类固醇异构酶不同，人类异构酶反应由二磷酸吡啶核苷酸（NADH，NAD+）刺激。亲和标记核苷酸类似物5′-[对-（氟磺酰基）苯甲酰基]腺苷（FSA）以相似的速率以不可逆的方式灭活脱氢酶和异构酶活性，其遵循关于时间和烷基化剂浓度（0.2-0.6 mM）的一级动力学。FSA是一种辅因子定点试剂，其结合亲和力与脱氢酶还原NAD+的竞争性抑制剂（Ki= 162 μM）或异构酶刺激剂（Km= 153 μM）相似。来自Kitz和Wilson分析的平行图表明，FSA以相等的烷基化效率（k3 Ki = 1/斜率= 0.5 l/mol-s）灭活两种酶活性。3β-羟基甾体底物，双烯醇酮，保护异构酶以及脱氢酶免于FSA失活。这些观察结果是一个单一的辅因子结合区，服务于两种酶的活动的证据。
3β-Hydroxy-5-ene-steroid dehydrogenase and steroid 5→4-ene-isomerase copurify as a single, homogeneous protein from human placental microsomes. Affinity alkylation with 2α-bromoacetoxyprogesterone suggests that the dehydrogenase and isomerase substrate steroids bind at different sites on the same protein. However, the coenzyme, NADH, completely abolishes the alkylation of both enzyme activities by the progestin analog [Thomas J. L., Myers R. P., Rosik L. O. and Strickler R. C., J. Steroid Biochem.36 (1990) 117–123]. Unlike bacterial 3-keto-5-ene-steroid isomerase, the human isomerase reaction is stimulated by diphosphopyridine nucleotides (NADH, NAD+). The affinity labeling nucleotide analog, 5′-[p-(fluorosulfonyl)benzoyl]adenosine (FSA), inactivates the dehydrogenase and isomerase activities at similar rates in an irreversible manner which follows first order kinetics with respect to both time and alkylator concentration (0.2–0.6 mM). FSA is a cofactor site-directed reagent that binds with similar affinity as a competitive inhibitor of NAD+reduction by dehydrogenase (Ki= 162 μM) or as a stimulator of isomerase (Km= 153 μM). Parallel plots derived from Kitz and Wilson analysis indicate that FSA inactivates the two enzyme activities with equal alkylation efficiency ( k3Ki= 1/ slope = 0.5 l/mol-s for both). The 3β-hydroxy-steroid substrate, pregnenolone, protects isomerase as well as dehydrogenase from inactivation by FSA. These observations are evidence for a single cofactor binding region which services both enzyme activities.