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Drug\u2010resistance demands exploring new targets against this illness. Methionyl\u2010tRNA synthetase (MetRS) is a crucial target in\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    <\/jats:styled-content>\n                    (\n                    <jats:italic>Mtb<\/jats:italic>\n                    ) that participates in the initiation and elongation of translation and represents a protein of evolutionary interest. To elucidate the structure\u2013function relationships of MetRS, we performed detailed sequence analyses and molecular dynamics simulations of\n                    <jats:italic>Mtb<\/jats:italic>\n                    MetRS in the substrate\u2010bound (methionine and ATP) and intermediate (methionyl\u2010AMP) states, for both the wild\u2010type and three single\u2010mutant forms (H21A, K54A, and E130A). Eight systems (two wild\u2010type and six mutants) were simulated for 36\u2009\u03bcs. Differential dynamics and binding effects of the substrate versus intermediate states were identified, along with the molecular reasons for the loss of activity in mutants. The wild\u2010type substrate state was more stable than the intermediate state. In contrast, the mutants were more unstable in the substrate state but incorporated stability into the intermediate state protein. These findings suggest that methionyl\u2010AMP, being a reaction intermediate, exhibits a short residence time at the protein's active site, while the substrate state shows a longer residence time of methionine and ATP. The increased instability of mutants in the substrate state indicates disruption of the pyrophosphate\u2010ATP exchange by altering substrate\u2013protein interactions. Once the intermediate is formed, the mutations have minimal or no effect. These observations are consistent with experimental data. In brief, our study finds the molecular basis for the distinct substrate and intermediate recognition by\n                    <jats:italic>Mtb<\/jats:italic>\n                    MetRS and establishes a mechanism for the loss of activity in the mutants.\n                  <\/jats:p>","DOI":"10.1002\/prot.70104","type":"journal-article","created":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T07:36:48Z","timestamp":1765784208000},"page":"1059-1073","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Distinct Substrate and Intermediate Recognition via Mutation Effects on\n                    <scp>\n                      <i>Mycobacterium tuberculosis<\/i>\n                    <\/scp>\n                    Methionyl\u2010\n                    <scp>tRNA<\/scp>\n                    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