STRUCTURAL ANALYSIS OF GLYCOCONJUGATE METABOLISM

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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.