DETAILED STUDIES ON SUBSTRATE STRUCTURE REQUIREMENTS OF GLYCOAMIDASE-A AND GLYCOAMIDASE-F

Glycoamidases eptide-N-4-(N-acetyl-beta-glucosaminyl)-asparagine amida se, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated beta-amide bond of the asparagine side ch ain. The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glyco amidase A or PNGase A). The study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides cont aining cellobiose, lactose, GlcNAc, and di-N,N'-acetylchitobiose (CTB) . The length of the peptide was varied from one to five amino acids, a nd glycosylamines were linked to either Asn or Gln located at differen t positions in the peptide, including NH2 and COOH termini. Neither en zyme could cleave cellobiose and lactose glycopeptides, indicating tha t the 2-acetamido group on the Asn-linked GlcNAc is important in the r ecognition by both glycoamidases A and F. GlcNAc peptides could be cle aved by both enzymes, albeit not as effectively as CTB glycopeptides. Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N -glycosylation) for activity. Glycoamidase A could even hydrolyze a Gl n-bound CTB glycopeptide, whereas the action of glycoamidase A could a ct on CTB dipeptides, glycoamidase A could act on CTB dipeptides, glyc oamidase F preferred a tripeptide or longer. The K-m and V-max values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 nM and 0.66 mu mol/min/mg, respectively. A natural glycodipeptide, Man (9)-GlcNAc(2)-Asn-Phe, was also completely hydrolyzed by glycoamidase A.