Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage.
N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.
D000117:Acetylglucosamine / Q000378:metabolism; D004187:Disaccharides; D005417:Flavobacterium / Q000201:enzymology; D005936:Glucans / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000378:metabolism*
D004187:Disaccharides; D005936:Glucans; D009844:Oligosaccharides; C032438:chitobiose; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D005990:Glycerol; D000117:Acetylglucosamine