Functional mimicry of the active site of glutathione peroxidase by glutathione imprinted selenium-containing protein.
Functional mimicry of the active site of glutathione peroxidase by glutathione imprinted selenium-containing protein.
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DOI：
10.1021/bm7008312
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发表时间：
2008
影响因子：
6.2
通讯作者：
Lei Liu;Shi-zhong Mao;Xiao-man Liu;Xin Huang;Jiayun Xu;Jun‐qiu Liu;G. Luo;Jia‐cong Shen
中科院分区：
文献类型：
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作者：
Lei Liu;Shi-zhong Mao;Xiao-man Liu;Xin Huang;Jiayun Xu;Jun‐qiu Liu;G. Luo;Jia‐cong Shen
为了模拟抗氧化剂硒酶谷胱甘肽过氧化物酶（GPx）的活性位点，采用人工酶selenosubtilisin作为支架，通过生物印迹策略重建底物谷胱甘肽（GSH）特异性结合位点。GSH首先通过Se-S键与selenosubtilisin共价连接，形成GSH-selenosubtilisin共价复合物，然后以GSH分子为模板，构建互补结合位点，用于底物GSH的识别。生物印迹过程包括展开selenosubtilisin的构象和固定复合物GSH-selenosubtilisin的新构象。从而获得了天然存在的GPx底物GSH的新特异性。这种生物印迹过程使印迹的selenosubtilisin的催化硒片段与GSH结合位点的活性巯基相匹配，有助于加速分子内催化。这些印迹含硒蛋白对GSH还原H2O2的速率显著提高。GPx的平均活性为462 U/微摩尔，约为未印迹硒枯草菌素的100倍。与著名的GPx模拟物ebselen相比，观察到活性增强了500倍。详细的稳态动力学研究表明，新型硒酶遵循类似于天然存在的GPx的乒乓机制。
For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.