Kong K H, Inoue H, Takahashi K
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Protein Eng. 1993 Jan;6(1):93-9. doi: 10.1093/protein/6.1.93.
The evolutionally conserved aspartyl residues (Asp57, Asp98 and Asp152) in human glutathione S-transferase P1-1 were replaced with alanine by site-directed mutagenesis to obtain the mutants (D57A, D98A and D152A). The replacement of Asp98 with alanine resulted in a decrease of the affinity for S-hexyl-GSH-agarose, a 5.5-fold increase of the KmGSH and a 2.9-fold increase of the I50 of S-hexyl-GSH for GSH-CDNB conjugation. Asp98 seems to participate in the binding of GSH through hydrogen bonding with the alpha-carboxylate of the gamma-glutamyl residue of GSH. The kcat of D98A was 2.6-fold smaller than that of the wild-type, and the pKa of the thiol group of GSH bound in D98A was approximately 0.8 pK units higher than those in the wild-type. Asp98 also seems to contribute to the activation of GSH to some extent. On the other hand, most of the kinetic parameters of D57A and D152A were similar to those of the wild-type. However, the thermostabilities of D57A and D152A were significantly lower than that of the wild-type. Asp57 and Asp152 seem to be important for maintaining the proper conformation of the enzyme.
通过定点诱变将人谷胱甘肽S-转移酶P1-1中进化保守的天冬氨酸残基（Asp57、Asp98和Asp152）替换为丙氨酸，以获得突变体（D57A、D98A和D152A）。将Asp98替换为丙氨酸导致对S-己基-GSH-琼脂糖的亲和力降低，KmGSH增加5.5倍，S-己基-GSH与GSH-CDNB结合的I50增加2.9倍。Asp98似乎通过与GSHγ-谷氨酰残基的α-羧酸盐形成氢键参与GSH的结合。D98A的kcat比野生型小2.6倍，D98A中结合的GSH硫醇基团的pKa比野生型高约0.8个pK单位。Asp98似乎在一定程度上也有助于GSH的活化。另一方面，D57A和D152A的大多数动力学参数与野生型相似。然而，D57A和D152A的热稳定性明显低于野生型。Asp57和Asp152似乎对维持酶的正确构象很重要。
