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STRUCTURAL ANALYSIS OF GLYCOCONJUGATE METABOLISM

糖复合物代谢的结构分析

基本信息

批准号：
6179720
负责人：
PATRICK M VAN ROEY
金额：
$ 16.5万
依托单位：
WADSWORTH CENTER
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 2002-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6179720
关键词：
N acetylglucosaminidase asparagine enzyme activity enzyme mechanism glycolipids glycoproteins lipid metabolism microorganism culture microorganism metabolism molecular cloning oligosaccharides protein metabolism recombinant proteins site directed mutagenesis

项目摘要

DESCRIPTION: The overall goal of this project is the analysis of the mechanism of action and substrate specificities of enzymes that cleave asparagine-linked oligosaccharides from glycoproteins. The study focuses on endoglycosidases and glycoamidases, enzymes that remove intact oligosaccharides. Endoglycosidases are glycohydrolases that cleave the glycosidic link between the two N-acetylglucosamine residues in the core of the oligosaccharide chain. Glycoamidases are amidohydrolases that convert asparagine residue to aspartic acid and remove the intact oligosaccharide. These enzymes are commonly used as biochemical tools for the analysis of glycoproteins and oligosaccharides. The three-dimensional structures of the enzymes and of complexes with substrates, products and inhibitors will be determined by x-ray crystallography, and analyzed in conjunction with site directed mutagenesis experiments to identify the residues involved in the mechanism of action and substrate binding. The project has three aims: (1) the analysis of the substrate specificities of four related endo-beta-N-acetylglucosaminidases, Endo H, Endo F1, Endo F2 and Endo F3 that have identical mechanisms but function on different asparagine-linked oligosaccharides; (2) the study of the mechanism of action and substrate recognition of two distinct types of glycoamidases, PNGase F and glycosylasparaginase, that catalyze the same reaction but have different structures, mechanisms and substrate requirements; and (3) the study of the mechanism of action and substrate specificity of Endo A, an endo-beta-N-acetylglucosaminidase that is unrelated to Endo H but has the same high-mannose substrate specificity, and that, unlike Endo H and related enzymes, has tranglycosylation activity. The results of these studies will be important for the development of better biochemical tools for the analysis of oligosaccharides, for the processing of glycoproteins and, in case of Endo A, for the modification of N-linked oligosaccharides of glycoproteins into forms suitable for therapeutic applications.

描述：本项目的总体目标是分析酶的作用机制和底物特异性来自糖蛋白的天冬酰胺连接的寡糖。研究的重点是内切糖苷酶和糖酰胺酶，去除完整的低聚糖。内切糖基化酶是切割糖基化酶的糖水解酶。核心中两个N-乙酰葡糖胺残基之间的糖苷键寡糖链。糖酰胺酶是酰胺水解酶，将天冬酰胺残基转化为天冬氨酸并除去完整的寡糖。这些酶通常被用作生化分析工具，糖蛋白和寡糖。的三维结构，酶和与底物、产物和抑制剂的复合物将是通过X射线晶体学测定，并结合研究中心进行分析定向诱变实验，以鉴定参与作用机制和底物结合。本项目有三个目标：（1）分析底物特异性四种相关的内切-β-N-乙酰氨基葡萄糖苷酶，Endo H，Endo F1，Endo F2 和Endo F3，它们具有相同的机制，但在不同的天冬酰胺连接寡糖;（2）作用机制的研究和两种不同类型的糖酰胺酶PNGase F的底物识别和糖基天冬酰胺酶，它们催化相同的反应，但具有不同的结构、机制和基底要求;和（3）研究 Endo A的作用机制和底物特异性，内切-β-N-乙酰氨基葡糖苷酶，与Endo H无关，但具有相同的高甘露糖底物特异性，并且与Endo H和相关酶，具有转糖基化活性。这些研究的结果将对于开发更好的生物化学工具，低聚糖分析，用于糖蛋白的加工，对于Endo A的情况，用于修饰N-linked寡糖，将糖蛋白转化成适合于治疗应用的形式。