N-acetylglucosaminidase cleaves all non-reducing terminal β-linked N-acetylglucosamine residues from complex carbohydrates and glycoproteins.
The cleavage rates of different linkages of GlcNAc on bi-, tri- and tetraantennary oligosaccharides is greatly dependent on the steric hindrance by neighbouring residues. The β(1-2)GlcNAc residue linked to the β(1-3)-linked mannose is cleaved at the highest rate and the β(1-2) GlcNAc residue linked to the β(1-6)-linked mannose at the lowest rate for all three oligosaccharides. The β(1-6) GlcNAc residue, when present, is removed at the second highest rate and the β(1-4) GlcNAc, third. On a triantennary structure, this residue is removed at the second highest rate. A bisecting β(1-4) GlcNAc linked to the β-linked mannose severely hinders cleavage of other GlcNAc residues–high concentrations of enzymes and prolonged incubation times are required for cleavage.