Ito N, Phillips S E, Stevens C, Ogel Z B, McPherson M J, Keen J N, Yadav K D, Knowles P F
Department of Biochemistry and Molecular Biology University of Leeds, UK.
Faraday Discuss. 1992(93):75-84. doi: 10.1039/fd9929300075.
Galactose oxidase is a copper-containing enzyme, which catalyses stereospecific oxidation of primary alcohols. The three-dimensional structure of the enzyme has been determined in this study by X-ray crystallography at high resolution. The molecule is almost entirely composed of beta-structures and consists of three domains. The arrangement of 28 beta-strands in the second domain is of particular interest, having seven four-stranded antiparallel beta-sheets with pseudo-sevenfold symmetry. The copper site has square-pyramidal coordination with two histidines, one tyrosine and one exogenous ligand at the equatorial sites and another tyrosine at the axial site. The most intriguing structural feature is a covalent bond between C epsilon 1 of Tyr-272, which is one of the equatorial ligands, and S gamma of Cys-228. This unexpected thioether bond, and Trp-290 stacked above it, strongly supports the presence of a tyrosine free radical in the enzyme as a 'built-in' secondary cofactor. Calculation of the molecular surface shows a small pocket at the copper site and suggests a substrate-binding model, which can explain the substrate specificity. A model for the catalytic mechanism, involving a tyrosine free radical and basic tryptophan, is also proposed.
半乳糖氧化酶是一种含铜酶，可催化伯醇的立体特异性氧化反应。本研究通过高分辨率X射线晶体学确定了该酶的三维结构。该分子几乎完全由β结构组成，包含三个结构域。第二个结构域中28条β链的排列方式特别有趣，具有七个具有伪七重对称性的四链反平行β折叠片层。铜位点具有四方锥配位结构，赤道面上有两个组氨酸、一个酪氨酸和一个外源性配体，轴向上有另一个酪氨酸。最引人注目的结构特征是作为赤道配体之一的Tyr-272的Cε1与Cys-228的Sγ之间存在共价键。这种意想不到的硫醚键以及位于其上方的Trp-290，有力地支持了该酶中存在酪氨酸自由基作为“内置”二级辅因子。分子表面计算显示铜位点处有一个小口袋，并提出了一个底物结合模型，该模型可以解释底物特异性。还提出了一种涉及酪氨酸自由基和碱性色氨酸的催化机制模型。
