高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (10): 2109.doi: 10.7503/cjcu20140101

• 有机化学 • 上一篇    下一篇

基于快速定点突变研究含硒人Zeta型谷胱甘肽硫转移酶1c-1c的催化活性

郭笑, 于扬, 陈龙, 王铭铄, 金梦梦, 刘广娜, 张黎, 李涛, 高壮, 魏景艳()   

  1. 吉林大学药学院, 长春 130021
  • 收稿日期:2014-02-11 出版日期:2014-10-10 发布日期:2014-09-18
  • 作者简介:联系人简介: 魏景艳, 女, 博士, 教授, 主要从事人工酶研究. E-mail: jingyanweijluedu@163.com
  • 基金资助:
    国家自然科学基金(批准号: 30970633, 31270851)、 吉林大学医学部博士研究生优秀拔尖人才培育计划(批准号: 470110000006)和大学生创新训练计划(批准号: 2013B73333)资助

Catalytic Activity of Seleno-hGSTZ1c-1c Based on Site-directed Mutagenesis

GUO Xiao, YU Yang, CHEN Long, WANG Mingshuo, JIN Mengmeng, LIU Guangna, ZHANG Li, LI Tao, GAO Zhuang, WEI Jingyan*()   

  1. College of Pharmaceutical Science, Jilin University, Changchun 130021, China
  • Received:2014-02-11 Online:2014-10-10 Published:2014-09-18
  • Contact: WEI Jingyan E-mail:jingyanweijluedu@163.com
  • Supported by:
    Supported by the National Natural Science Foundation of China(Nos.30970633, 31270851), the Doctoral Funding Grants of Norman Bethune Health Science Center of Jilin University, China(No.470110000006) and the Undergraduate Innovative Training Program of China(No.2013B73333)

摘要:

先将人Zeta型谷胱甘肽硫转移酶1c-1c(hGSTZ1c-1c)中非催化中心的Cys-137, Cys-154, Cys-165和Cys-205突变为Ser, 然后将催化中心14, 15和17位的3个氨基酸残基突变为Cys, 再利用半胱氨酸缺陷型大肠杆菌表达系统将其特定地转化为Sec, 即把GPx的催化基团引入到hGSTZ1c-1c中, 高效地获得了具有谷胱甘肽过氧化物酶(GPx)活力的模拟酶. 其中制备的3个含硒突变体15C, 14C/15C和17C均显示出明显的GPx活力. 对非含硒突变体性质研究发现, Ser-14或Ser-15任何一个残基发生突变都会导致hGSTZ1c-1c的GST活力几乎丧失, 表明Ser-14和Ser-15在催化反应中发挥着重要作用, 但前者主要参与底物结合, 后者更侧重于催化.

关键词: 快速定点突变, 谷胱甘肽过氧化物酶, Zeta型谷胱甘肽硫转移酶

Abstract:

Human glutathione transferase zeta 1c-1c(hGSTZ1c-1c) is considered to be an ideal protein scaffold for imitating glutathione peroxidase(GPx) owing to the natural binding site of glutathione(GSH). In this research, four cysteine(Cys) residues(Cys-137, Cys-154, Cys-165 and Cys-205) were mutated to serine(Ser) to avoid untargeted introduction of selenocysteine(Sec) residues, which could lead to structural change. Then Ser-14, Ser-15 and Ser-17 near the GSH binding site were mutated to Cys, respectively, and biosynthetically converted to Sec by a Cys auxotrophic expression system. Of those mutants, the seleno-containing mutants 15C, 14C/15C and 17C showed some GPx activities. Substitution of either Ser-14 or Ser-15 resulted in the loss of GST acitivity of hGSTZ1c-1c, indicating that Ser-14 and Ser-15 may play crucial roles in the reaction. And subsequent study suggested that Ser-14 may be essential in binding of GSH, while Ser-15 was probably involved in catalysis.

Key words: Site-directed mutagenesis, Glutathione peroxidase, Human glutathione transferase zeta

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