Resonance energy transfer between sites in rat liver glutathione S-transferase, 1-1, selectively modified at cysteine-17 and cysteine-111.
Resonance energy transfer between sites in rat liver glutathione S-transferase, 1-1, selectively modified at cysteine-17 and cysteine-111.
复制标题
大鼠肝脏谷胱甘肽 S-转移酶 1-1 位点之间的共振能量转移，在半胱氨酸 17 和半胱氨酸 111 处进行选择性修饰。
DOI：
10.1021/bi962119j
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发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
Colman,RF
中科院分区：
文献类型：
--
作者：
Hu,L;Colman,RF
Monobromobimane (mBBr) can label both Cys111and Cys17of rat liver glutathioneS-transferase, 1-1 (GST 1-1). However, selective modification of Cys111was achieved by the maleimide-based sulfhydryl reagentsN-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 °C leads to the formation of modified enzyme with 92% residual activity toward 1-chloro-2,4-dinitrobenzene and completely blocks Cys111from 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 Cys17with loss of activity. Together with the only tryptophan (Trp20), fluorescein linked to Cys111and bimane to Cys17provide three fluorescent probes to study the solution structure of GST 1-1. Fluorescence spectral analysis suggests that Trp20and bimane linked to Cys17are located in a relatively hydrophobic environment, while fluorescein linked to Cys111is 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 Å between Trp20and bimane at Cys17, 19 Å between Trp20and fluorescein at Cys111, and <22 Å between bimane at Cys17and 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.
