The action of N-acetylglucosaminyltransferase-V is prevented by the bisecting GlcNAc residue at the catalytic step

Using a purified protein and bisected acceptor oligosaccharides, we demonstrate that N-acetylglucosaminyltransferase (GnT)-V transfers a N-acetylglucosamine residue via a β1,6-linkage to the bisected oligosaccharides. We also kinetically characterized the substrate specificity of GnT-V with respect to the bisected oligosaccharide. Although the K m values for the bisected acceptors were comparable to that for a non-bisected acceptor, the V max values for the bisected acceptors were much lower than that for the non-bisected acceptor. These findings suggest that the acceptor specificity of GnT-V is determined by the catalytic process rather than by its binding to the substrate. It was also found that the presence of the 2- N-acetyl group in the bisecting monosaccharide moiety plays a critical role in determining the catalytic efficiency of the enzyme.