STRUCTURAL ANALYSIS OF GLYCOCONJUGATE METABOLISM

糖复合物代谢的结构分析

• 批准号: 2188282
• 负责人: PATRICK M VAN ROEY
• 金额: $ 13.18万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2188282
• 关键词: N acetylglucosamine; N acetylglucosaminidase; X ray crystallography; amidases; computer program /software; computer simulation; crystallization; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; fluorescent dye /probe; glycopeptides; glycoproteins; mutant; polysaccharides; site directed mutagenesis; structural biology; synthetic peptide

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.

该项目的目标是分析作用机制和 代谢糖复合物的酶的底物特异性。这 研究重点是对结构要求的调查 通过糖苷内切酶水解 N-连接多糖 糖肽酰胺酶。四个endo-(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 酰胺酶，N4-(N-乙酰基-(3-D-氨基葡萄糖)天冬酰胺酶(PNGase F)， 将通过X射线晶体学分析来测定。四个 糖苷内切酶裂解两个 N- 之间的 β(l-4) 连接 N-连接的 N,N'-二乙酰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特异性 结构：高甘露糖（Endo H 和 Endo F1）、双触角（Endo F2）和 三触角（Endo 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。 糖苷内切酶需要 N,N'-二乙酰壳二糖核心并且至少 三个额外的特定碳水化合物残基用于活性。 PNG酶F 需要 N,N'-二乙酰壳二糖核心和肽骨架。 结构研究将解释这些大识别的必要性 网站。 酶与底物和产物的复合物的结构 将确定类似物。定点诱变将用于 产生具有改变的活性和/或特异性的酶，包括 可以结合但不能加工糖肽的突变体。结构性 对这些突变体与完整底物的复合物进行分析将提供 完整详细地了解作用机制和 这些酶的结合特异性。 了解多糖如何被识别以及多糖的含量有多高 区分甘露糖、杂合和复杂结构非常重要 用于研究多糖在各种物质中的功能 生物过程，主要是细胞间糖蛋白的识别 相互作用和受体结合。潜在的长期应用 这些研究的结果将用于开发这些酶或 突变体作为生产特定糖型的工具 用于治疗用途的糖蛋白和多糖。