摘要
用1-乙基-3-(3-二甲基氨基丙基)-碳二亚胺(EDC),2.4.6.三硝基苯磺酸(TNBS)和丁二酮(DIC)分别修饰人胎盘型谷胱甘肽S-转移酶(GST-π)的羧基、氨基和胍基,研究了酶的失活动力学,发现引起一分子酶亚基全部失活所需抑制剂的分子数分别为1.0、1.08和0.98,提示每亚基只有一个羧基、氨基和胍基参与酶的活性中心。底物及其类似物谷胱甘肽,S-己烷或S-辛烷谷胱甘肽可保护GST-π免受上述抑制剂的修饰,使假一级反应速度常数k_1明显降低,说明羧基、氨基和胍基是GST-π和GSH结合部位的组成基团。作者曾证明GST-π中的一个快反应巯基也参与酶与GSH的结合,故至少有四个不同的基团是酶亚基的结合基团,本文还对TNBS对氨基修饰的特异性作了验证,并讨论了GST-π与GSH结合时形成离子键的情况。
By using l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) ,2,4,6-trinitrobenzylsulfonic acid and diacetyl for the chemical modification of carboxyl,amino and guanido group of human glutathione S-transferase (GST-π) respectively to study the kinetics for inactivation of enzyme revealed that the corresponding moles of inhibitor needed for fully inactivation of the enzyme were 1.0, 1.08, 0.98/enzyme subunit, suggusting that only one mole of carboxyl, amino and quanido group for each participated in the active center of GST-π subunit. The substrate and its analogs, glutatione, S-hexylglutathione and S-octylglutathione, protected GST-π from modification by the above inhibitors and the rate constant of pseudo-first order reaction k1 was remarkably decreased in the presence of these protectors, indicting that carboxyl, amino and guanido groups were involved in the GSH binding site of GST-π. The authors had proved that one fast-reacting sulfhydryl group also participated in the binding of GST-π with GSH , so that at least 4 different groups were the binding groups of each subunit. The specific modification of amino group by TNBS was also verified and the ionic linkages between GST-π and GSH were proposed.
关键词
谷胱甘肽
S-转移酶
化学修饰
羧基
Glutathione S-transferase
Chemical modification
Carboxyl group
Amino group
Guanido group
