Structural and catalytic role of two conserved tyrosines in Delta-class glutathione S-transferase from Locusta migratoria.
Structural and catalytic role of two conserved tyrosines in Delta-class glutathione S-transferase from Locusta migratoria.
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DOI：
10.1002/arch.21025
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发表时间：
2012-07
影响因子：
2.2
通讯作者：
Xueyao Zhang;Tao Li;Jianqin Zhang;Daqi Li;Ya-ping Guo;Guohua Qin;Kun Yan Zhu;E. Ma;Jianzhen Zhan
中科院分区：
文献类型：
--
作者：
Xueyao Zhang;Tao Li;Jianqin Zhang;Daqi Li;Ya-ping Guo;Guohua Qin;Kun Yan Zhu;E. Ma;Jianzhen Zhan
谷胱甘肽S-转移酶（GSTs）是一类重要的解毒酶家族，在昆虫的抗药性中起着关键作用。酪氨酸是其解毒功能所必需的。在本研究中，两个保守的酪氨酸残基位于位置108和116的LmGSTD 1的H-位点。为了阐明这两个残基如何参与催化过程并保持结构稳定性，产生了四个突变体，Y108 A，Y108 E，Y116 A和Y116 E。结果表明，这4种突变体对LmGSTD 1的比活性有不同程度的影响，这取决于底物的种类和反应机理。稳态动力学分析表明，Y108 E和Y116 E对GSH结合能力有显著影响，说明H位的两个酪氨酸残基参与了G位的拓扑重排。Y116 A和Y116 E均表现出较低的CDNB结合亲和力，表明Y116参与疏水底物结合。热稳定性测定、固有荧光和8-苯胺基-1-萘磺酸（ANS）荧光结果表明，这两个酪氨酸残基参与活性位点构象的调节。最后，同源性建模提供了证据表明，在H-位的两个酪氨酸参与疏水底物结合。此外，Y108更接近S-己基谷胱甘肽的S原子。总之，LmGSTD 1中的两个酪氨酸是催化过程和蛋白质稳定性中的重要残基。
Glutathione S-transferases (GSTs) are an important family of detoxifying enzymes and play a key role in pesticide resistance in the insect. Tyrosine is essential for its detoxification function. In the present study, two conserved tyrosine residues are located at positions 108 and 116 in H-site of LmGSTD1. To elucidate how the two residues participate in the catalytic process and keeping structural stability, four mutants, Y108A, Y108E, Y116A, and Y116E, were generated. It was found that the four mutants affected the specific activity of LmGSTD1 in various degrees, depending on the types of substrate and reaction mechanism. Steady-state kinetics assay revealed that Y108E and Y116E had a significant influence on GSH-binding ability, which indicates the two tyrosine residues of H-site contribute to topology rearrangement of G-site. Both Y116A and Y116E exhibited lower CDNB-binding affinity, suggesting that Y116 takes part in hydrophobic substrate binding. The thermostability assay, intrinsic, and 8-anilino-1-naphthalenesulfonic acid (ANS) florescence results showed that the two tyrosine residues were involved in regulation of active-site conformation. Finally, homology modeling provided evidence that the two tyrosines in H-site participate in hydrophobic substrate binding. Furthermore, Y108 is closer to the S atom of S-hexylglutathione. In conclusion, the two tyrosines in LmGSTD1 are important residues in both the catalytic process and protein stability.