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Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate.

ABSTRACT: Trypanosoma cruzi trans-sialidase (TcTS) is a crucial enzyme for the infection of Trypanosoma cruzi, the protozoa responsible for Chagas' disease in humans. It catalyzes the transfer of sialic acids from the host's glycoconjugates to the parasite's glycoconjugates. Based on kinetic isotope effect (KIE) studies, a strong nucleophilic participation at the transition state could be determined, and recently, elaborate experiments used 2-deoxy-2,3-difluorosialic acid as substrate and were able to trap a long-lived covalent intermediate (CI) during the catalytic mechanism. In this paper, we compute the KIE and address the entire mechanistic pathway of the CI formation step in TcTS using computational tools. Particularly, the free energy results indicate that in the transition state there is a strong nucleophilic participation of Tyr342, and after this, the system collapsed into a stable CI. We find that there is no carbocation intermediate for this reaction. By means of the energy decomposition method, we identify the residues that have the biggest influence on catalysis. This study facilitates the understanding of the catalytic mechanism of TcTS and can serve as a guide for future inhibitor design studies.

SUBMITTER: Pierdominici-Sottile G

PROVIDER: S-EPMC3779645 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate.

Pierdominici-Sottile Gustavo G Horenstein Nicole A NA Roitberg Adrian E AE

Biochemistry 20111027 46

Trypanosoma cruzi trans-sialidase (TcTS) is a crucial enzyme for the infection of Trypanosoma cruzi, the protozoa responsible for Chagas' disease in humans. It catalyzes the transfer of sialic acids from the host's glycoconjugates to the parasite's glycoconjugates. Based on kinetic isotope effect (KIE) studies, a strong nucleophilic participation at the transition state could be determined, and recently, elaborate experiments used 2-deoxy-2,3-difluorosialic acid as substrate and were able to tra ...[more]

PMID: 22007596

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Project description:Oral transmission of T. cruzi is probably the most frequent mechanism among animals in the wild. In this context, there is a high prevalence of human infections in regions where triatomine infection with T. cruzi is low. This led to the hypothesis that the consumption of raw or undercooked meat from animals infected with T. cruzi could be responsible for the transmission of the infection. Therefore, the general objective of this study was to demonstrate the role of meat consumption from infected animals in the oral transmission of T. cruzi infection. Groups of five female mice Balb/c were fed with muscles obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacity. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies. In all cases, 60–100% of animals were fed meat from mice infected in the acute phase or infected by means of a nasogastric probe with trypomastigotes or amastigotes. These animals developed high parasitemia, and 80% died around day 40 post-infection. The adhesion tests showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS also confirmed that transialidase may be involved in TCT attachment of TCT or invasion of stomach cells. It was concluded that the consumption of meat from infected animals in the acute phase of T. cruzi infection allows transmission of the infection. Similarly, trypomastigotes and amastigotes were able to infect mice when administered orally, while cruzipain was a relevant molecule in this infective process.

Project description: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 ?-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 ?-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.

Dataset Information

Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate.

Publications

Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate.

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