Hara T, Tanaka T, Kato H, Nishioka T, Oda J
Institute for Chemical Research, Kyoto University, Japan.
Protein Eng. 1995 Jul;8(7):711-6. doi: 10.1093/protein/8.7.711.
Lys18, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathione synthetase from Escherichia coli B are presumed to be highly concerned with the substrate, gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), binding by X-ray crystallography and affinity labeling studies. Using site-directed mutagenesis, we investigated functional roles of those residues for gamma-Glu-Cys binding. The mutant enzymes of Arg86 and Asn283 altered their kinetic parameters, especially the Michaelis constants of gamma-Glu-Cys. In the case of Asn283, the residue is not likely to have an essential role in gamma-Glu-Cys binding but its side chain would extend to make a van der Waals contact with bound gamma-Glu-Cys. Chemical modification of a cysteine residue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86 would not only be much responsible for gamma-Glu-Cys binding but would also have a role in maintaining the structural integrity of the enzyme. The other mutant enzymes showed little defect in their kinetic parameters of gamma-Glu-Cys.
通过X射线晶体学和亲和标记研究推测，来自大肠杆菌B的谷胱甘肽合成酶的Lys18、Arg86、Asn283、Ser286、Thr288和Glu292与底物γ-L-谷氨酰-L-半胱氨酸（γ-Glu-Cys）的结合高度相关。我们使用定点诱变研究了这些残基在γ-Glu-Cys结合中的功能作用。Arg86和Asn283的突变酶改变了它们的动力学参数，特别是γ-Glu-Cys的米氏常数。就Asn283而言，该残基在γ-Glu-Cys结合中不太可能起关键作用，但其侧链会延伸以与结合的γ-Glu-Cys形成范德华接触。用5,5'-二硫代双（2-硝基苯甲酸）（DTNB）对一个半胱氨酸残基进行化学修饰表明，Arg86不仅对γ-Glu-Cys结合起很大作用，还在维持酶的结构完整性方面发挥作用。其他突变酶在γ-Glu-Cys的动力学参数上几乎没有缺陷。
