Asp-AMS is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10 nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). Asp-AMS has also the highest inhibitory effect for the mitochondrial enzyme. Asp-AMS is the most active inhibitor with Ki values in the nanomolar range, with a stronger effect on bacterial AspRSs (E. coli and P. aeruginosa) than on human cytosolic AspRS.