STRUCTURAL ANALYSIS OF GLYCOCONJUGATE METABOLISM

这个项目的目标是分析作用机制和 代谢糖共轭化合物的酶的底物专一性。这个 研究的重点是调查结构的要求 糖苷内切酶对N-连接多糖的水解性裂解 糖肽酰胺酶。四个内酯-(3-N-)的三维结构 乙酰氨基葡萄糖苷酶、Endo H、Endo F1、Endo F2和Endo F3以及 N4-(N-乙酰-(3-D-氨基)氨基)天冬酰胺酰胺酶(PNGase F)， 将通过X射线结晶学分析确定。四个 内切糖苷酶切断两个N-之间的β-(L-4)连接 N-链N，N‘-二乙酰基壳二糖核心中的乙酰氨基葡萄糖 多糖，但对不同的多糖具有专一性 结构：高甘露糖(Endo H和Endo F1)，双天线(Endo F2)和 三天线(远藤F3)。PNGase F去除完整的N-连接 多糖链，并将天冬酰胺残基转化为天冬氨酸。 内切糖苷酶需要N，N‘-二乙酰基壳二糖核心和至少 三个额外的特定碳水化合物残留物的活性。PNGase F 需要N，N‘-二乙酰基壳二糖核心和多肽骨架。 结构研究将解释这些大的认可的必要性 网站。 酶与底物及产物的络合物结构 类似物将被确定。定点突变将被用于 产生活性和/或特异性改变的酶，包括 可以结合但不能处理糖肽的突变体。结构性的 对这些突变体与完整底物的复合体的分析将提供 完全详细地了解行动机制和 这些酶的结合专一性。 对多糖是如何识别的以及多糖的含量有多高的理解。 甘露糖、杂交糖和复杂结构的区分具有重要意义 为了研究不同种类的多糖的功能 生物过程，主要识别细胞-细胞中的糖蛋白 相互作用和受体结合。潜在的长期应用 这些研究的结果将在这些酶的开发或 突变体作为生产特定糖型的工具 治疗用糖蛋白和多糖类。

The goal of this project is the analysis of the mechanisms of action and substrate specificity of enzymes that metabolize glycoconjugates. The study focusses on the investigation of the structural requirements for the hydrolytic cleavage of N-linked polysaccharides by endoglycosidases and glycopeptide amidases. The three-dimensional structures of four endo-(3-N- acetylglucosaminidases, Endo H, Endo F1, Endo F2 and Endo F3, and the amidase, N4-(N-acetyl-(3-D-glucosaminyl)asparagine amidase (PNGase F), will be determined by X-ray crystallographic analysis. The four endoglycosidases cleave the beta(l-4)link between the two N- acetylglucosamines in the N,N'-diacetylchitobiose core of N-linked polysaccharides but with specificity for distinct polysaccharide structures: high-mannose (Endo H and Endo F1), biantennary (Endo F2) and triantennary (Endo F3). PNGase F removes the intact N-linked polysaccharide chain and converts the asparagine residue to an aspartate. The endoglycosidases require the N,N'-diacetylchitobiose core and at least three additional specific carbohydrate residues for activity. PNGase F requires both the N,N'-diacetylchitobiose core and the peptide backbone. The structural studies will explain the need for these large recognition sites. The structures of complexes of the enzymes with substrate and product analogues will be determined. Site directed mutagenesis will be used to produce enzymes with altered activity and/or specificity, including mutants that can bind but not process glycopeptides. The structural analysis of complexes of these mutants with intact substrates will provide complete detailed understanding of the mechanism of action and of the binding specificity of these enzymes. The understanding of how polysaccharides are recognized and how high- mannose, hybrid and complex structures are distinguished is of importance for the study of the functionality of polysaccharides in various biological processes, primarily recognition of glycoproteins in cell-cell interaction and receptor binding. Potential long term application of the results of these studies will be in the development of these enzymes or mutants as tools for the production of specific glycoforms of glycoproteins and of polysaccharides for therapeutic use.