Trypanosoma cruzitrans-sialidase (TcTS), which catalyzes the transfer or hydrolysis of terminal sialic acid residues, is crucial to the development and proliferation of the T. cruzi parasite and thus has emerged as a potential drug target for the treatment of Chagas disease. We here probe the origin of the observed preference for the transfer reaction over hydrolysis where the substrate for TcTS is the natural sialyl donor (represented in this work by sialyllactose). Thus, acceptor lactose preferentially attacks the sialyl-enyzme intermediate rather than water. We compare this with the weaker preference for such transfer shown by a synthetic donor substrate, 4-methylumbelliferyl α-d-acetylneuraminide. For this reason, we conducted molecular dynamics simulations of TcTS following its sialylation by the substrate to examine the behavior of the asialyl leaving group by the protein. These simulations indicate that, where lactose is released, this leaving group samples well-defined interactions in the acceptor site, some of which are mediated by localized water molecules; also, the extent of the opening of the acceptor site to solvent is reduced as compared with those of unliganded forms of TcTS. However, where there is release of 4-methylumbelliferone, this leaving group explores a range of transient poses; surrounding active site water is also more disordered. The acceptor site explores more open conformations, similar to the case in which the 4-methylumbelliferone is absent. Thus, the predicted solvent accessibility of sialylated TcTS is increased when 4-methylumbelliferyl α-d- acetylneuraminide is the substrate compared to sialyllactose; this in turn is likely to contribute to a greater propensity for hydrolysis of the covalent intermediate. These computational simulations, which suggest that protein flexibility has a role in the transferase/sialidase activity of TcTS, have the potential to aid in the design of anti-Chagas inhibitors effective against this neglected tropical disease. © 2013 American Chemical Society.