STRUCTURAL ANALYSIS OF GLYCOCONJUGATE METABOLISM

描述：本项目的总体目标是分析 裂解酶的作用机制和底物特异性 糖蛋白中的天冬酰胺连接低聚糖。这项研究的重点是 内切糖苷酶和糖酰胺酶，这些酶可以完整地去除 低聚糖。内切糖苷酶是一种糖水解酶，可以裂解 核心区两个N-乙酰氨基葡萄糖残基之间的糖苷连接 低聚糖链。糖酰胺酶是一种氨基水解酶，可以将 将天冬酰胺残留物转化为天冬氨酸，并除去完整的寡糖。 这些酶通常被用作生化工具来分析 糖蛋白和低聚糖。的三维结构 酶以及与底物、产物和抑制剂的络合物 由x射线结晶学测定，并结合现场分析 定向突变实验，以确定参与的残基 作用机制和底物结合。 该项目有三个目标：(1)底物特性分析 四种相关的内切β-N-乙酰氨基葡萄糖苷酶：内切H、内切F1、内切F2 和Endo F3具有相同的机制，但在不同的 天冬酰胺连接的寡糖；(2)作用机理的研究 和底物识别两种不同类型的糖胺酶，PNGase F 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、机制和衬底要求；以及(3)对 Endo A，An的作用机制和底物专一性 内切-β-N-乙酰氨基葡萄糖苷酶与内切H无关，但具有 相同的高甘露糖底物专一性，而不像Endo H和相关 酶，具有三角糖基化活性。这些研究的结果将 对开发更好的生物化学工具具有重要意义 低聚糖的分析，用于糖蛋白的加工和，在 Endo A案，用于修饰N-连接的低聚糖 将糖蛋白转化为适合治疗应用的形式。

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.