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.