Hu L, Colman R F
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Biochemistry. 1997 Feb 18;36(7):1635-45. doi: 10.1021/bi962119j.
Monobromobimane (mBBr) can label both Cys111 and Cys17 of rat liver glutathione S-transferase, 1-1 (GST 1-1). However, selective modification of Cys111 was achieved by the maleimide-based sulfhydryl reagents N-ethylmaleimide (NEM) and fluorescein 5-maleimide (NFM). Incubation of GST 1-1 with 5 mM NEM for 30 min at pH 7.5 and 25 degrees C leads to the formation of modified enzyme with 92% residual activity toward 1-chloro-2,4-dinitrobenzene and completely blocks Cys111 from subsequent reaction with either NFM or mBBr. Reaction of GST 1-1 with 0.2 mM NFM under the same conditions affords a modified enzyme with only 14% residual activity even though NFM and NEM target the same Cys111. The results indicate that when the bulky fluorescein is covalently bound to Cys111, the ligand projects into both the xenobiotic binding site and the glutathione site. After NEM or NFM modification of GST 1-1, the enzyme was further modified by monobromobimane at Cys17 with loss of activity. Together with the only tryptophan (Trp20), fluorescein linked to Cys111 and bimane to Cys17 provide three fluorescent probes to study the solution structure of GST 1-1. Fluorescence spectral analysis suggests that Trp20 and bimane linked to Cys17 are located in a relatively hydrophobic environment, while fluorescein linked to Cys111 is located in a charged environment. These fluorescent probes constitute three sets of donor-acceptor pairs for the measurement of fluorescence energy transfer, and distances calculated from such measurements are 20 A between Trp20 and bimane at Cys17, 19 A between Trp20 and fluorescein at Cys111, and < 22 A between bimane at Cys17 and fluorescein at Cys111. Molecular modeling studies indicate that fluorescein lies between the two subunits, is surrounded by charged residues, and is extended into the xenobiotic binding site. They also suggest that mBBr must approach from the dimer interface in order to reach the reaction site at Cys17.
单溴联亚胺（mBBr）可以标记大鼠肝脏谷胱甘肽S-转移酶1-1（GST 1-1）的Cys111和Cys17。然而，基于马来酰亚胺的巯基试剂N-乙基马来酰亚胺（NEM）和荧光素5-马来酰亚胺（NFM）实现了对Cys111的选择性修饰。在pH 7.5和25℃条件下，将GST 1-1与5 mM NEM孵育30分钟，会形成对1-氯-2,4-二硝基苯具有92%残余活性的修饰酶，并完全阻止Cys111与NFM或mBBr发生后续反应。在相同条件下，GST 1-1与0.2 mM NFM反应，即使NFM和NEM靶向相同的Cys111，也会得到仅具有14%残余活性的修饰酶。结果表明，当庞大的荧光素与Cys111共价结合时，配体既伸入外源性物质结合位点，也伸入谷胱甘肽位点。用NEM或NFM对GST 1-1进行修饰后，该酶在Cys17处被单溴联亚胺进一步修饰，活性丧失。与唯一的色氨酸（Trp20）一起，连接到Cys111的荧光素和连接到Cys17的联亚胺提供了三个荧光探针来研究GST 1-1的溶液结构。荧光光谱分析表明，与Cys17相连的Trp20和联亚胺位于相对疏水的环境中，而与Cys111相连的荧光素位于带电环境中。这些荧光探针构成了三组供体-受体对用于测量荧光能量转移，通过这种测量计算出的距离为：Trp20与Cys17处的联亚胺之间为20 Å，Trp20与Cys111处的荧光素之间为19 Å，Cys17处的联亚胺与Cys111处的荧光素之间小于22 Å。分子模拟研究表明，荧光素位于两个亚基之间，被带电荷的残基包围，并延伸到外源性物质结合位点。研究还表明，mBBr必须从二聚体界面靠近才能到达Cys17处的反应位点。
