Identification of two histidyl residues in the active site of human placental estradiol 17 beta-dehydrogenase.
Identification of two histidyl residues in the active site of human placental estradiol 17 beta-dehydrogenase.
复制标题
人胎盘雌二醇 17 β-脱氢酶活性位点中两个组氨酰残基的鉴定。
DOI：
10.1021/bi00257a012
复制
发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Warren,JC
中科院分区：
文献类型：
--
作者：
Chin,CC;Murdock,GL;Warren,JC
作者：加里L.默多克，** 和詹姆斯C. Warren摘要：为了进一步表征人胎盘雌二醇17/3-脱氢酶（EC 1.1. 1.62），合成了12/3-（溴乙酰氧基）-4-雌烯-3，17-二酮和16 α-（溴乙酰氧基）-1，3，5（10）-雌三烯-3，17 β-二醇3-甲醚。12/3衍生物的合成步骤包括4-雌烯-3，17-二酮被胶孢炭疽菌特异性地12/3-羟基化，然后与溴乙酸偶联。这两种亲和标记类固醇都是底物，并以时间依赖性、不可逆的方式抑制酶，雌二醇-17/3使其减慢。均相酶样品（4 μ）分别用400 μ 12/3-（溴[2- 14 C]乙酰氧基）-4-雌烯-3，17-二酮和16 α-（溴[2- 3 H]乙酰氧基）-1，3，5（10）-雌三烯-3，17/3-二醇3-甲基醚在pH 6.3下灭活。酸水解后，这些样品的等分试样均产生放射性1-（羧甲基）组氨酸、3-（羧甲基）组氨酸、1，3-双（羧甲基）组氨酸、5-（羧甲基）半胱氨酸和少量（羧甲基）赖氨酸。在失活过程中，雌二醇-17/3的存在减缓了组氨酰烷基化（在3位最显著），但对半胱氨酸的烷基化几乎没有影响，
Chang-Chen Chin, Gary L. Murdock,** and James C. Warren abstract: To further characterize the active site of human placental estradiol 17/3-dehydrogenase (EC 1.1. 1.62), we have synthesized 12/3-(bromoacetoxy)-4-estrene-3, l7-dione and 16a-(bromoacetoxy)-1, 3, 5 (10)-estratriene-3, 17/S-diol 3-methyl ether. Synthetic steps for the 12/3 derivative involved specific12/3-hydroxylation of 4-estrene-3, 17-dione by Collectotrichum gloeosporioides and subsequent coupling with bromoacetic acid. Both of the affinity-labeling steroids are substrates and inactivate the enzyme in a time-dependent, irreversible manner, which is slowed by estradiol-17/3. Samples of homogeneous enzyme (4 µ) were inactivated separately with 400 µ 12/3-(bromo [2-14C] acetoxy)-4-estrene-3, 17-dione and 16a-(bromo [2-3H] acetoxy)-l, 3, 5 (10)-estratriene-3, 17/3-diol 3-methyl ether at pH 6.3. Aliquots of these samples, after acid hydrolysis, both yielded radioactive l-(carboxymethyl) histidine, 3-(carboxymethyl) histidine, 1, 3-bis (carboxymethyl) histidine, 5-(carboxymethyl) cysteine, and small amounts of (carboxymethyl) lysine. The presence of estradiol-17/3 during the inactivation slowed histidyl alkylation (most significantly at the 3 position) but had little effect on alkylation of cysteine by