INTERACTION OF CHITOBIOSE WITH PNGASE F MUTANTS. Peter Kuhn, SSRL, Stanford, CA 94309, USA and Patrick Van Roey, Wadsworth Center, NYSDOH, Albany, NY 122201, USA

Peptide-N4-(N-acetylglucosaminyl)asparagine amidase F (PNGase F) is an amidohydrolase secreted by Flavobacterium meningosepticum. The enzyme releases intact Asn-linked oligosaccharides from glycoproteins and glycopeptides while converting the asparagine residue to an aspartic acid. Based on crystallographic and mutagenesis studies, three acidic residues, Asp60, Glu118 and Glu206, were shown to be important for catalytic activity or substrate binding. All three residues are found in a cleft at the interface between the two [[beta]]-sandwich domains of the enzyme. Asp60 and Glu206 are located at one end of the cleft and are connected by a bridging water molecule. Glu118 is located towards the other end of the cleft. The structures of the amide mutants of all three residues, Asn60, Gln118 and Gln206, are essentially identical to that of the wild-type enzyme. This proves that the reduced activities of the mutants, less than 0.01% of the wild-type enzyme, result from the chemical modification and not from a structural effect. The structures of the wild-type enzyme and all three mutants have also been determined using crystals that were co-crystallized with 30-fold excess of N,N'-diacetylchitobiose, the Asn-proximal core of the oligosaccharide product. The structural analysis shows that the disaccharide binds well to the wild-type enzyme and to the Gln206 mutant, less well to the Asn60 mutant and not at all to the Gln118 mutant. These results are consistent with the location of the disaccharide in the binding site: O1 of the first GlcNAc forms a hydrogen bond with Asp60, while O6 of the second GlcNAc is in hydrogen bonding contact with Glu118. Therefore, Asp60 is most likely the primary catalytic residue, while Glu206 plays a secondary in catalytic process. Glu118 is essential for substrate binding but not involved in the catalytic mechanism..