J. Biol. Chem., Vol. 260, Issue 29, 15488-15494, 12, 1985
1H NMR evidence that almond "peptide: N-glycosidase" is an amidase. Kinetic data and trapping of the intermediate
JM Risley and RL Van Etten
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.
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