Eukaryotes have distinct nuclear genes for tryptophanyl-tRNA synthetase (TrpRS) enzymes targeted by N-terminal sequence variations to the cytoplasm (Hc) and mitochondria (Hmt) that share only 14% sequence identity. Indolmycin, a natural tryptophan analog, competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Although bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan, KM ~2 μM, eukaryotic cytosolic TrpRS enzymes are able to evade inhibition by indolmycin. Tryptophan binding to Bacillus stearothermophilus (Bs) TrpRS is largely promoted by hydrophobic interactions and recognition of the indole nitrogen by the side chain of Asp 132. By contrast, HcTrpRS complements non polar interactions for tryptophan binding with electrostatic and hydrogen bonding interactions, which we show by modelling are inconsistent with indolmycin binding. Our crystallographic and inhibition kinetics data show the non-reactive analog indolmycin can recruit unique polar interactions to form an active-site metal coordination that lies off the normal mechanistic path, enhancing affinity to BsTrpRS and other prokaryotic TrpRS enzymes by 1500-fold over its tryptophan substrate. The Mg2+ ion in the inhibited complex forms significantly closer contacts with triphosphate oxygen atoms of ATP and three water molecules than occur in the catalytically-competent pre-transition state (preTS). Indolmycin binding also leads to weakened interactions between ATP and active-site lysine side-chains. Confirmation of our interpretation of structural consequences of indolmycin binding comes from a 1.82 Å crystal structure of an indolmycin-inhibited HmtTrpRS complex. This structure unequivocally demonstrates the use of similar determinants by mitochondrial and bacterial TrpRS enzymes to bind both ATP and indolmycin, with the mitochondrial enzyme forming similar ATP-enzyme, ATP-metal and indolmycin-enzyme interactions. Indolmycin binds HmtTrpRS ~700-times tighter than tryptophan and Mg2+•ATP leads to an ~80-fold enhancement in indolmycin binding affinity. The oxazolinone- Mg2+•ATP interaction contributes ~-2.2 kcal/mol to the Gibbs free energy of the fully-liganded indolmycin inhibited HmtTrpRS complex. Together, our complementary structural, kinetic and thermodynamic characterization of BsTrpRS and HmtTrpRS establish a shared mechanism for indolmycin inhibition of mitochondrial and prokaryotic TrpRS enzymes, which bind indolmycin 105-106-fold tighter than eukaryotic cytosolic homologs. Advisors/Committee Members: Williams, Tishan, Carter, Charles, Ke, Hengming, Beese, Lorena, Fried, Howard.