Baron A J, Stevens C, Wilmot C, Seneviratne K D, Blakeley V, Dooley D M, Phillips S E, Knowles P F, McPherson M J
Department of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
J Biol Chem. 1994 Oct 7;269(40):25095-105.
Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site.
对半乳糖氧化酶的晶体学和光谱学研究表明，活性位点涉及酪氨酸272上的一个自由基，酪氨酸272是与Cu2+辅因子配位的配体之一。酪氨酸272与半胱氨酸228之间存在一种新型硫醚键，并且色氨酸290堆积在该键上方，这些都是晶体结构的特征。本研究描述了半乳糖氧化酶高水平异源表达系统的开发以及这些关键活性位点残基处突变变体的构建。通过X射线晶体学、可见光谱学和催化活性测量对表达的野生型酶和突变变体（W290H和C228G）进行了表征。另一种变体蛋白Y272F无法纯化。数据表明硫醚键和堆积的色氨酸对活性至关重要，并进一步支持色氨酸290作为自由基位点组成部分的作用。
