N-Acetylglucosamine beta-O-linked to serine and threonine residues of nucleocytoplasmic proteins (O-GlcNAc) has been linked to neurodegeneration, cellular stress response. and transcriptional regulation Removal of O-GlcNAc is catalyzed by O-GlcNAcase (OGA) Using, a substrate-assisted catalytic mechanism. Here we define the reaction coordinate Using chemical approaches and directly observe both a Michaelis complex and the oxazoline intermediate.