A Neutral Diphosphate Mimic Crosslinks the Active Site of Human O-GlcNAc Transferase

Glycosyltransferases (Gtfs) catalyze the formation of a diverse array of glycoconjugates. Small molecule inhibitors to manipulate Gtf activity in cells have long been sought as tools to understand Gtf function. Success has been limited due to challenges in designing inhibitors that mimic the negatively-charged diphosphate substrates. Here we report the mechanism of action of a small molecule that inhibits O-GlcNAc transferase (OGT), an essential human enzyme that modulates cell signaling pathways by catalyzing a unique intracellular post translational modification, β-O-GlcNAcylation. The molecule contains a five heteroatom dicarbamate core that functions as a neutral diphosphate mimic. One dicarbamate carbonyl reacts with an essential active site lysine that anchors the diphosphate of the nucleotide-sugar substrate. The lysine adduct reacts again with a nearby cysteine to crosslink the OGT active site. While this unprecedented mechanism reflects the unique architecture of the OGT active site, related dicarbamate scaffolds may inhibit other enzymes that bind diphosphate containing substrates.