Time-dependent, reversible, oxaborole inhibition of Escherichia coli leucyl-tRNA synthetase measured with a continuous fluorescence assay

Enzyme assays for the catalytic activity of aminoacyl-tRNA synthetases generally measure the incorporation of radioactive amino acids into tRNA. Such assays are necessarily discontinuous. Leucyl-tRNA synthetase has recently gained attention as the target of novel antimicrobial compounds based on the oxaborole scaffold, examples of which have been shown to have slow binding and dissociation kinetics. Investigations of the kinetics of inhibition by these compounds would be facilitated by a continuous assay of leucyl-tRNA synthetase catalysis. Here we report a continuous fluorescence intensity-based assay for leucyl-tRNA synthetase in which the pyrophosphate product is converted to phosphate, which is detected with nanomolar sensitivity by a phosphate sensor protein. This assay was used to measure the time constants for the slow onset of inhibition and long residence time of an oxaborole-based inhibitor.