Small molecule covalent modification of proteins has proven valuable in activity-based protein profiling (ABPP) studies to probe enzyme mechanisms and creating potent inhibitors. While several electrophiles exhibit high selectivity for nucleophilic residues such as cysteine and serine, the identification of electrophiles to specifically target lysine residues has been challenging. Due to the natural abundance of lysine residues, lysine modification could offer new insight into enzyme structure and mechanisms. However this ubiquity has complicated the production of highly selective and stable compounds. Squaric esters possess unique reactivity making them suitable candidates as novel electrophilic warheads for selectively modifying lysine residues. Using galactofuranosyltransferase 2 (GlfT2), a key enzyme in <i>M. tuberculosis</i> cell wall biosynthesis, as our model system, we showed that substrate analogs containing a squaric ester moiety covalently inhibited GlfT2. Mass spectrometry pinpointed the covalent modification and showed evidence of covalent binding near the active site as well as another lysine more distal to the active site. This modification was conserved across two other homologs of GlfT2 revealing a potential allosteric binding pocket. These results highlight the utility of using squaric esters as lysine specific electrophiles for designing covalent probes to study enzyme structure and dynamics.

Squaric esters as novel lysine specific electrophiles

256th National Meeting (2018)

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