Abstract
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.
Highlights
Trypanosoma cruzi trans-sialidase (TcTS) is a potential target for Chagas disease chemotherapy
Our results show remarkable structural plasticity upon engagement of acceptor substrate in the TcTS active site, a property that must be considered in future studies on the catalytic mechanism of the enzyme and in the design of new selective inhibitors
Lac binding reduced Asp-59 solvation in the TS ternary complex (Fig. 6C), which suggests that entry of the acceptor substrate displaced water molecules from the catalytic site and supported the high rates of transfer reaction exhibited by TS
Summary
Trypanosoma cruzi trans-sialidase (TcTS) is a potential target for Chagas disease chemotherapy. Based on structural [11] and kinetic studies [14], Tyr-342 is considered to be the catalytic nucleophile residue of TcTS [11], whereas Asp-59 is proposed to act as a general acid/base catalyst in a double displacement reaction [14] that follows a classical ping-pong mechanism [15] in which the sialosyl-aglycone may leave the active site to allow entry of an acceptor substrate According to this hypothesis, a water molecule could attack the sialosyl-enzyme intermediate before the acceptor substrate reaches the binding site, which would result in hydrolysis rather than in an efficient sugar transfer. Our results show remarkable structural plasticity upon engagement of acceptor substrate in the TcTS active site, a property that must be considered in future studies on the catalytic mechanism of the enzyme and in the design of new selective inhibitors
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