Human placental estradiol 17 beta-dehydrogenase: sequence of a histidine-bearing peptide in the catalytic region.
Human placental estradiol 17 beta-dehydrogenase: sequence of a histidine-bearing peptide in the catalytic region.
复制标题
人胎盘雌二醇 17 β-脱氢酶：催化区域中带有组氨酸的肽的序列。
DOI：
10.1021/bi00351a019
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发表时间：
1986
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：
Murdock,GL;Chin,CC;Warren,JC
人胎盘雌二醇17/3-脱氢酶（EC 1.1. 1.62）通过亲和标记技术建立。在pH6.3时，分别用12/3-羟基-4-雌烯-3，17-二酮12-（溴[2- 14 C]乙酸酯）和3-甲氧基雌三醇16-（溴[2- 14 C]乙酸酯）灭活该酶。灭活，在这两种情况下，遵循伪一级动力学与半衰期为12/3和16 a衍生物分别为192和68小时。这两种衍生物都是已知的底物，其以时间依赖性、不可逆的方式降解，并修饰半胱氨酸残基以形成（羧甲基）半胱氨酸和组氨酸残基以形成NT-或AT-（羧甲基）组氨酸。失活的酶样品分别进行还原、羧甲基化和胰蛋白酶消化。将胰蛋白酶消化物应用于Sephadex G-50，鉴定了带有放射性AT-和A”-（羧甲基）组氨酸的肽。通过阳离子交换层析和凝胶过滤进一步纯化肽。在测序之前通过HPLC实现最终纯化。已确定两种类固醇衍生物修饰肽Thr-Asp-Ile-His-Thr-Phe-His-Arg中的两个组氨酸残基中的任一个。这些组氨酸不同于以前被雌酮3-（溴乙酸酯）烷基化的组氨酸，并且推测其接近结合类固醇的A环。结论：本研究中鉴定的两个组氨酸残基与甾体D环的活性位点结合，可能参与人胎盘雌二醇17/3-脱氢酶（EC 1.1. 1.62）先前已纯化至均质（Chin & Warren，1973）并结晶（Chin等人，1976年，在这个实验室里。活性部位的地形已被研究了各种类固醇溴乙酸酯的亲和标记。类固醇轴承
The amino acid sequence of an octapeptide from the catalytic site of human placental estradiol 17/3-dehydrogenase (EC 1.1. 1.62) was established by affinity-labeling techniques. The enzyme was inactivated separately by 12/3-hydroxy-4-estrene-3, 17-dione 12-(bromo [2-14C] acetate) and 3-methoxyestriol 16-(bromo [2-14C] acetate) at pH 6.3. The inactivations, in both cases, followed pseudo-first-order kinetics with half-times for the 12/3 and 16a derivatives being 192 and 68 h, respectively. Both derivatives are known substrates that inactivate in a time-dependent, irreversible manner and that modify cysteine residues to form (carboxymethyl) cysteine and histidine residues to form either NT-or AT-(carboxymethyl) histidine. The inactivated enzymesamples were separately reduced, carboxymethylated, and digested with trypsin. The tryptic digests were applied to Sephadex G-50 and the radioactive AT-and A”-(carboxymethyl) histidine-bearing peptides identified. The peptides were further purified by cation-exchange chromatography and gel filtration. Final purification was achieved by HPLC prior to sequencing. It was determined that both steroid derivatives modified either of the two histidine residues in the peptide Thr-Asp-Ile-His-Thr-Phe-His-Arg. These histidines are different from a histidine that was previously shown to be alkylated by estrone 3-(bromoacetate) and that was presumed to proximate the A ring of the bound steroid. It is concluded that thetwo histidine residues identified in thepresent study proximate the D ring of the steroid as it binds at the active site and may participate in the hydrogen transfer effected by human placental estradiol 17/3-dehydrogenase.Estradiol 17/3-dehydrogenase (EC 1.1. 1.62) has previously been purified to homogeneity (Chin & Warren, 1973) and crystallized (Chin et al., 1976) in this laboratory. The topography of the active site has been studied by affinity labeling with various steroid bromoacetates. Steroids bearing the