Trypanosoma cruzi trans-sialidase (TcTS) is a key target protein for Chagas disease chemotherapy. In this study, we investigated the implications of active site flexibility on the biochemical mechanism of TcTS. Molecular dynamics studies revealed remarkable plasticity in the TcTS catalytic site, demonstrating, for the first time, how donor substrate engagement with the enzyme induces an acceptor binding site in the catalytic pocket that was not previously captured in crystal structures. Furthermore, NMR data showed cooperative binding between donor and acceptor substrates, supporting theoretical results. In summary, our data put forward a coherent dynamic framework to understand how a glycosidase evolved its highly efficient trans-glycosidase activity.