Kong K H, Inoue H, Takahashi K
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
J Biochem. 1992 Dec;112(6):725-8. doi: 10.1093/oxfordjournals.jbchem.a123965.
The four residues of human glutathione S-transferase P1-1 whose counterparts were indicated by X-ray crystallography to reside in the GSH-binding site of pig glutathione S-transferase P1-1 were individually replaced with threonine or alanine by site-directed mutagenesis to obtain mutants R13T, K44T, Q51A, and Q64A. The kinetic parameters, susceptibilities to an inhibitor, S-hexyl-GSH, and affinities for GSH-Sepharose of the latter were compared with those of the wild-type enzyme, and pKa of the thiol group of GSH bound in R13T was shown to be equivalent to that in the wild type. From the results, Lys44, Gln51, and Gln64 were deduced to contribute to the binding of GSH. On the other hand, Arg13 seems to be essential for the enzymatic activity as mainly involved in the construction of a proper structure of the active site.
通过X射线晶体学表明，猪谷胱甘肽S-转移酶P1-1的谷胱甘肽（GSH）结合位点中的对应残基，与人谷胱甘肽S-转移酶P1-1的四个残基分别通过定点诱变被苏氨酸或丙氨酸取代，从而获得突变体R13T、K44T、Q51A和Q64A。将后者的动力学参数、对抑制剂S-己基-GSH的敏感性以及对GSH-琼脂糖的亲和力与野生型酶进行了比较，结果表明R13T中结合的GSH硫醇基团的pKa与野生型中的相当。从结果推断，Lys44、Gln51和Gln64有助于GSH的结合。另一方面，Arg13似乎对酶活性至关重要，因为它主要参与活性位点正确结构的构建。
