In plants and microorganisms the key aromatic compounds involved in primary metabolism, including the three aromatic amino acids, are produced by the shikimate pathway. Animals, in contrast, have to obtain their aromatic compounds from their diet. Thus, there is a keen interest in the shikimate-pathway enzymes as potential targets for the development of non-toxic herbicides and antimicrobial compounds. The 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), the sixth enzyme in this pathway, has been recognized as a potential target for development of herbicides and antibiotic drugs. The 3D structure of this enzyme was determined and glyphosate identified as a potent inhibitor for a series of EPSPS of different origins, including that of M. tuberculosis (Mt). This work aims to study the conformational dynamics of the inactive and active forms of the MtEPSPS by using the hydrogen amide exchange (HX), mass spectrometry (MS) and circular dichroism. The results showed that the binding of inhibitor resulted in increasing of the structural organization, characterized by conformational changes from the active to the inactive MtEPSPS forms. A comparison in the deuterium incorporation of the pepsic peptides from the MtEPSPS in presence and absence of the inhibitor revealed a more extensive deuteration of the active enzyme, suggesting a decrease of solvent accessibility to the glyphosate-bound enzyme when compared to the free enzyme. Some regions showed significant differences in deuterium incorporation, specially those containing the amino acids residues of the glyphosate binding site (fragments 16-32; 112-121; 121-127; 195-205; 335-343; 382-390). It was also demonstrated that these fragments belong to a-helices regions that suffer extensive conformational changes during the inhibitor binding, in order to optimize the enzyme-inhibitor interactions. These results also demonstrate the capacity of HX/MS for examining the conformational changes of proteins dynamics.