1H NMR evidence that almond "peptide: N-glycosidase" is an amidase. Kinetic data and trapping of the intermediate.
The enzyme from almond that catalyzes the hydrolysis of the N-glycosidic linkage between asparagine and the oligosaccharide chain of glycopeptides and glycoproteins has been variously termed an N-glycosidase and an amidase enzyme. Using turkey ovomucoid glycopeptide as a substrate for the enzyme, we followed the hydrolysis reaction by 1H NMR spectroscopy. These kinetic data revealed a rapid hydrolysis of the substrate but a delayed appearance of the final product. This implied that an intermediate, most likely a 1-aminooligosaccharide, was formed during the reaction. Identification of the intermediate as a 1-beta-amino-N-acetylglucosamine-oligosaccharide was achieved by trapping it as the 1-acetamido derivative using acetic anhydride and subsequent analysis by 1H NMR. The data conclusively demonstrate that the enzyme catalyzes the hydrolysis of the glycopeptide to form an aspartic acid-containing polypeptide and an intermediate oligosaccharide amine. The latter derivative is hydrolyzed nonenzymatically to yield the final carbohydrate product. Thus, the enzyme is in fact an amidohydrolase (amidase) and not an N-glycosidase. The trivial name glycopeptidylamidase is suggested.
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D009754:Nuts; D010059:Ovomucin / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D014422:Turkeys
D010059:Ovomucin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase