Wang J, Barycki J J, Colman R F
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Protein Sci. 1996 Jun;5(6):1032-42. doi: 10.1002/pro.5560050606.
Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.
大鼠肝脏谷胱甘肽S-转移酶同工酶1-1与异源生物底物类似物4-（氟磺酰基）苯甲酸（4-FSB）反应，在pH 6.5条件下以1-氯-2,4-二硝基苯（CDNB）为底物进行测定时，会导致该酶随时间失活，最终活性降至其原始活性的35%。失活速率对4-FSB浓度在0.25 mM至9 mM范围内呈非线性依赖，其特征为抑制常数（KI）为0.78 mM，最大失活速率（kmax）为0.011 min-1。S-己基谷胱甘肽或异源生物底物类似物2,4-二硝基苯酚可保护该酶不被4-FSB失活，而S-甲基谷胱甘肽对该反应影响很小。这些实验表明反应发生在酶的活性位点内，可能在异源生物底物的结合位点，靠近谷胱甘肽结合位点。在不存在和存在S-己基谷胱甘肽的情况下，将[3,5-3H]-4-FSB掺入酶中表明，一个残基的修饰导致了酶活性的部分丧失。已证明Tyr 8和Cys 17是4-FSB的反应靶点，但只有Tyr 8能被S-己基谷胱甘肽保护免受4-FSB作用。二硫苏糖醇（DTT）可从半胱氨酸与4-FSB的反应产物中再生半胱氨酸，但不能使酶重新激活。这些结果表明，4-FSB对Tyr 8的修饰导致了酶的部分失活。酶对各种底物的米氏常数不会因酶的修饰而改变。与天然酶相比，修饰酶催化谷胱甘肽与CDNB反应的pH依赖性表明，表观pKa增加了约0.9，这被解释为代表酶结合谷胱甘肽的电离；然而，修饰酶的这个约7.4的pKa仍远低于游离谷胱甘肽-SH的9.1的pK。以前，人们认为Tyr 8对谷胱甘肽S-转移酶催化至关重要。相反，我们得出结论，Tyr 8促进了与谷胱甘肽S-转移酶结合的谷胱甘肽硫醇基团的电离，但不是酶活性所必需的。
