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.

该项目的目标是分析作用机制， 代谢糖缀合物的酶的底物特异性。的 研究的重点是调查的结构要求， 通过内切糖苷酶水解裂解N-连接的多糖， 糖肽酰胺酶。四种endo-（3-N- 乙酰氨基葡糖苷酶，Endo H，Endo F1，Endo F2和Endo F3，以及 酰胺酶，N4-（N-乙酰基-（3-D-葡糖胺基）天冬酰胺酰胺酶（PNGase F）， 将通过X射线晶体学分析确定。四 内切糖基化酶切割两个N-糖基化酶之间的β（1 -4）连接。 N，N '-二乙酰基壳二糖核心中的乙酰葡糖胺 但对不同多糖具有特异性 结构：高甘露糖（Endo H和Endo F1），双触角（Endo F2）和 三触角（Endo F3）。PNGase F去除了完整的N-连接的 在一个实施方案中，所述方法包括将所述多糖链与天冬酰胺残基结合并将天冬酰胺残基转化为天冬氨酸。 内切糖苷酶需要N，N '-二乙酰壳二糖核心，并且至少 三个额外的特定碳水化合物残基的活性。PNGase F 需要N，N ′-二乙酰基壳二糖核心和肽骨架。 结构研究将解释这些大的识别的需要 网站. 酶与底物和产物复合物的结构 将确定类似物。定点诱变将用于 产生活性和/或特异性改变的酶，包括 可以结合但不加工糖肽的突变体。结构性 这些突变体与完整底物的复合物的分析将提供 完整详细的了解作用机制和 这些酶的结合特异性。 对多糖是如何被识别的以及有多高的理解- 区分甘露糖、杂合结构和复杂结构是重要的 用于研究多糖在各种 生物过程，主要是识别细胞-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， 突变体作为生产特定糖型的工具， 糖蛋白和多糖用于治疗用途。