SUBSTRATE RECOGNITION BY ENZYMES THAT RELEASE OLIGOSACCHARIDES FROM GLYCOPROTEINS. Patrick Van Roey, Wadsworth Center, New York State Dept. of Health, Albany, NY 12201-0509, USA

Flavobacterium meningosepticum secretes four oligosaccharide releasing enzymes: three glycohydrolases, endo-(-N-acetylglucosaminidase (Endo) F₁, F₂ and F₃, and the amidohydrolase, peptide-N-(N-acetyl-(-D-glucosaminyl)asparagine amidase (PNGase) F. The enzymes remove asparagine-linked oligosaccharides and are used as biochemical tools for the analysis of glycoproteins. All four enzymes have unique substrate specificities. PNGase F removes the intact oligosaccharide chain and converts the asparagine to an aspartic acid. The minimum substrate for PNGase F consists of the asparagine residue with both the carboxyl and amino groups in peptide linkage and the chitobiose core of the oligosaccharide. Endo F₁, F₂ and F₃, as well as the related Endo H, cleave the ((1-4)-glycosidic bond between the two N-acetylglucosamines of the chitobiose core. They differ strongly in their respective specificities for different oligosaccharide structures: F₁ (and H), high-mannose; F₂, biantennary; and, F₃, triantennary. Crystallographic studies of the enzymes, mutants and complexes are aimed at the analysis of the mechanisms of action and the basis for the substrate specificities of the enzymes. PNGase F is composed of two 8-stranded (-sandwich domains that are positioned side-by-side. Loops that connect the (-strands form a cleft at the interface between the two domains. Site directed mutagenesis studies combined with crystallographic analysis of the chitobiose complex have shown that this cleft contains the active site residues and the oligosaccharide binding site. The endoglycosidases are ((/()₈-barrels. The structures of Endo F₁ and H reveal distinct features associated with the recognition of the branched high-mannose chain, the difference in tolerance for an ((1-3)-fucose on the asparagine-proximal N-acetylglucosamine and the interaction with the protein component of the glycoprotein substrate.