A Novel Lactose-Sensitive Switch Mechanism Inactivates T. cruzi Trans-sialidase´s Covalent Intermediate, as Revealed by Molecular Dynamics Studies

DIEGO ALONSO DE ARMIÑO; DARÍO A. ESTRIN; ALVAREZ, ROSA MARÍA S.; ADRIÁN ROITBERG

Tucuman

Congreso; XLI Reunión Anual de la ociedad Argentina de Biofísica; 2012

Sociedad Argentina de Bofísica

Trypanosoma cruzi is the causative agent of Chagas´ disease, endemic in South and Central America. Despite being the cause of thousands of death annually, the chronic form has no effective treatment to date. T. cruzi trans-sialidase (TcTS) was shown to be an important factor in the microorganism´s virulence, and has been proposed as a target for drug design, a goal that requires a through understanding of the enzyme´s mechanism which, in spite of great advances in the past decades, is still incomplete. Evidence indicates that the catalytic mechanism involves a long-lived covalent intermediate (CI). A key unanswered question is thus, how does TcTS protect the CI from hydrolysis before the acceptor glicoconjugate can possition itself in the active site for the transfer reaction to take place?. Here we deal with this problem by comparing the stability of the reactive configuration in the CI form of TcTS with that of Tripanosoma Rangeli Sialidase (TrSA) (a structurally and mechanistically very similar enzyme with strict hydrolase activity) using molecular dynamics simulations. Unbiased molecular dynamics show a diminished population of the reactive configuration of the CI-TcTS compared to CI-TrSA in absence of an acceptor ligand (i.e. lactose). Also, umbrella sampling studies show striking differences between the free energy surfaces of CI-TcTS, CI-TcTS bound to lactose, and CI-TrSA, which indicate a lactose-sensitive mechanism for inactivation of the enzyme in the CI stage.