STRUCTURAL ANALYSIS OF GLYCOCONJUGATE METABOLISM

糖复合物代谢的结构分析

• 批准号: 2188283
• 负责人: PATRICK M VAN ROEY
• 金额: $ 11.53万
• 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/2188283
• 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.

该项目的目的是分析行动机制和 代谢糖缀合物的酶的底物特异性。这 研究的重点是研究的结构要求 N连接多糖的水解切割，由内糖苷酶和 糖肽胺。四个内部的三维结构（3-n-） 乙酰葡萄糖氨基酶，Endo H，Endo F1，Endo F2和Endo F3，以及 amidase，N4-（N-乙酰基 - （3-D-葡萄糖胺基）天冬酰胺酰胺酶（PNGase F），F） 将通过X射线晶体学分析确定。四个 内糖苷酶在两个N-之间切割β（L-4）链接 N连接的N，N'-diacetylchitobiose核心中的乙酰葡萄糖胺 多糖，但对于不同的多糖具有特异性 结构：High-Mannose（Endo H和Endo F1），Biantennary（Endo F2）和 Triantennary（Endo F3）。 PNGase F删除完整的N连接 多糖链并将天冬酰胺残基转换为天冬氨酸。 内糖苷酶需要N，N'DiacetylChitobiose核心，至少需要 三个其他特定的碳水化合物残基用于活性。 pngase f 同时需要N，N'-二乙基氯二烯核心和肽主链。 结构研究将解释对这些庞大认识的需求 站点。 酶与底物和产物的复合物结构 将确定类似物。定位的诱变将用于 产生具有改变活性和/或特异性的酶，包括 可以结合但不处理糖肽的突变体。结构 对这些突变体具有完整底物的复合物的分析将提供 完全详细了解行动机制和 这些酶的结合特异性。 对如何识别多糖的理解以及有多高 区分甘露糖，混合和复杂的结构是重要的 为了研究各种多糖的功能 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 这些研究的结果将是这些酶的开发或 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。