INACTIVATION OF STEROID SULFATASE BY AN ACTIVE-SITE-DIRECTED INHIBITOR, ESTRONE-3-O-SULFAMATE
INACTIVATION OF STEROID SULFATASE BY AN ACTIVE-SITE-DIRECTED INHIBITOR, ESTRONE-3-O-SULFAMATE
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DOI：
10.1021/bi00036a025
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发表时间：
1995-09-12
期刊：
影响因子：
2.9
通讯作者：
REED, MJ
中科院分区：
文献类型：
--
作者：
PUROHIT, A;WILLIAMS, GJ;REED, MJ
类固醇硫酸酯酶负责水解3 β-羟基类固醇硫酸酯，例如胆固醇和硫酸双烯醇酮，并且在调节内分泌依赖性肿瘤中由硫酸雌酮和硫酸脱氢表雄酮合成雌激素类固醇中具有重要作用。虽然很少有人知道的机制，硫酸基团被删除的类固醇核，一个积极的定点硫酸酯酶抑制剂已被开发。以雌酮的钠盐为原料，经氨磺酰氯处理，合成了雌酮-3-O-氨基磺酸酯（EMATE）。该化合物不仅抑制雌酮硫酸酯酶，而且抑制胎盘微粒体和完整MCF-7乳腺癌细胞中的脱氢表雄酮硫酸酯酶活性。用EMATE预处理MCF-7细胞或胎盘微粒体，然后进行大量洗涤或透析，表明不可逆抑制。EMATE以时间、浓度和pH依赖性方式抑制胎盘微粒体中的雌酮硫酸酯酶活性，证实了这一点。该酶被硫酸雌酮保护免于失活，这也与活性位点定向抑制一致。EMATE被提议通过酶的不可逆氨磺酰化作用来抑制雌酮硫酸酯酶。在pH 8.6时检测到最大酶活性，EMATE的最大酶失活速率也发生在该pH。酶促反应的pK（a）值和失活的pK（a）值分别为7.2和9.8，这证明两个活性位点残基被EMATE修饰。由于酪氨酸的酚pK（a）（9.7）和组氨酸的pK（a）（6.8）与失活的pK（a）值相似，因此这些氨基酸残基可能在催化机制中发挥作用。类固醇硫酸酯酶活性的活性位点定向的不可逆抑制剂的发展将允许这些酶在许多生理和病理过程中的作用进行评估，也有助于确定这种酶的活性位点。
Steroid sulfatases are responsible for the hydrolysis of 3 beta-hydroxy steroid sulfates, such as cholesterol and pregnenolone sulfate, and have an important role in regulating the synthesis of estrogenic steroids, from estrone sulfate and dehydroepiandrosterone sulfate, in endocrine-dependent tumors. Although little is known about the mechanism by which the sulfate group is removed from a steroid nucleus, an active site-directed sulfatase inhibitor has been developed. This inhibitor, estrone-3-O-sulfamate (EMATE), was synthesized by treating the sodium salt of estrone with sulfamoyl chloride. This compound inhibited not only estrone sulfatase but also dehydroepiandrosterone sulfatase activity in placental microsomes and in intact MCF-7 breast cancer cells. Pretreatment of MCF-7 cells or placental microsomes with EMATE, followed by extensive washing or dialysis indicated irreversible inhibition. This was confirmed by showing that EMATE inhibited estrone sulfatase activity in placental microsomes in a time-, concentration-, and pH-dependent manner. The enzyme is protected from inactivation by estrone sulfate, which is also consistent with active site-directed inhibition. EMATE is proposed to inactivate estrone sulfatase by irreversible sulfamoylation of the enzyme. Maximum enzyme activity was detected at pH 8.6, and the maximum rate of enzyme inactivation by EMATE also occurred at this pH. The pK(a) values of the enzymatic reaction and pK(a) of inactivation were 7.2 and 9.8, providing evidence that two active site residues are being modified by EMATE. As the phenolic pK(a) of tyrosine (9.7) and the pK(a) of histidine (6.8) are similar to the pK(a) values of inactivation, these amino acid residues may play a role in the catalytic mechanism. The development of an active site-directed irreversible inhibitor of steroid sulfatase activity will allow the roles that these enzymes have in a number of physiological and pathological processes to be evaluated and also help to identify the active site of this enzyme.