【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.更多还原

【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 15 C,14C /15 C and 17 C 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.更多还原