Structure and binding properties of hen lysozyme modified at tryptophan 62.
Structure and binding properties of hen lysozyme modified at tryptophan 62.
复制标题
色氨酸 62 修饰的母鸡溶菌酶的结构和结合特性。
DOI：
10.1016/0022-2836(81)90080-2
复制
发表时间：
1981
影响因子：
5.6
通讯作者：
Wilson,KS
中科院分区：
文献类型：
--
作者：
Blake,CC;Cassels,R;Dobson,CM;Poulsen,FM;Williams,RJ;Wilson,KS
本文用核磁共振氢谱和X射线晶体学研究了N-溴代琥珀酰亚胺修饰的鸡蛋清溶菌酶衍生物的结构3.2.1.17结果表明，将色氨酸残基改变为羟吲哚丙氨酸†残基的这种修饰仅在修饰位点引起微小的结构变化，并且天然酶的整体结构在衍生物中得以保持。根据先前的观察结果，通过所采用的两种方法观察到羟吲哚丙氨酸-62溶菌酶的两种非对映体（Norton & Allerhand，1976）。Glu 35和Asp 52的催化重要羧基的pK值在天然酶及其衍生物中是相同的。然而，修饰的酶在水解溶壁微球菌的细胞壁粘多糖中几乎是无活性的。本文用核磁共振波谱法研究了N-乙酰氨基葡萄糖寡糖与天然溶菌酶和Ox-62溶菌酶的结合，观察了溶菌酶~ 1H n.m.r.共振，和两个系统的扰动的差异表明，结合（GlcNAc）3特别是在两个系统中是不相同的。通过X射线晶体学研究Ox-62溶菌酶-（GlcNAc）3的结构，并且显示仅两个GlcNAc残基与酶接触，以与GlcNAc的α-异头形式结合天然酶类似的模式结合还原性末端残基（Blakeet al.，1967年a）。根据X射线晶体学和~ 1H n.m.r.在光谱中，Ox-62溶菌酶的酶活性的缺乏是由于活性位点的亚位点B的羟吲哚丙氨酸残基的阻碍，阻止了底物的生产性结合。
The structure of a derivative of hen egg-white lysozyme (EC 3.2.1.17) modified byN-bromosuccinimide at Trp62 has been studied by both1H nuclear magnetic resonance spectroscopy and X-ray crystallography. It was shown that this modification, changing the tryptophan residue to an oxindolealanine†residue, only causes minor structural changes at the site of the modification, and that the overall structure of the native enzyme is maintained in the derivative. Both diastereomers of the oxindolealanine-62 lysozyme were observed by the two methods employed, in accordance with previous observations (Norton & Allerhand, 1976). The pKvalues of the catalytically important carboxyl groups of Glu35 and Asp52 were identical in the native enzyme and its derivative. However, the modified enzyme is virtually inactive in the hydrolysis of the cell-wall mucopolysaccharide ofMicrococcus lysodeikticus. The binding ofN-acetylglucosamine oligosaccharides to both native lysozyme and Ox-62 lysozyme was studied by nuclear magnetic resonance spectroscopy, observing the perturbations on the lysozyme1H n.m.r. resonances, and differences in the perturbations of the two systems demonstrated that binding of (GlcNAc)3in particular was not identical in the two systems. The structure of Ox-62 lysozyme-(GlcNAc)3was studied by X-ray crystallography and it was shown that only two GlcNAc residues make contact with the enzyme, binding the reducing end residue in a similar mode as the α-anomeric form of GlcNAc binds to the native enzyme (Blakeet al., 1967a). On the basis of the results obtained by X-ray crystallography and1H n.m.r. spectroscopy, the lack of enzymatic activity of the Ox-62 lysozyme arises from the obstruction by the oxindolealanine residue of sub-site B of the active site, preventing productive binding of the substrate.