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Proton transfer facilitated by ligand binding. An energetic analysis of the catalytic mechanism of Trypanosoma cruzi trans-sialidase.

ABSTRACT: Trans-sialidase is a crucial enzyme for the infection of Trypanosoma cruzi, the protozoa responsible for Chagas' disease in humans. This enzyme catalyzes the transfer of sialic acids from mammalian host cells to parasitic cell surfaces in order to mask the infection from the host's immune system. It represents a promising target for the development of therapeutics to treat the disease and has been subject of extensive structural studies. Elaborate experiments suggested formation of a long-lived covalent intermediate in the catalytic mechanism and identified a Tyr/Glu pair as an unusual catalytic couple. This requires that the tyrosine hydroxyl proton is transferred to the carboxylate group of glutamate before the nucleophilic attack. Since the solution pK(a)s of tyrosine and glutamate are very different, this transfer can only be accomplished if the reaction environment selectively stabilizes the product state. We compute the free energy profile for the proton transfer in different environments, and our results indicate that it can take place in the active site of trans-sialidase, but only after substrate binding. By means of the energy decomposition method, we explain the influence that the active site residues exert on the reaction and how the pattern is changed when the substrate is present. This study represents an initial step that can shed light on our understanding of the catalytic mechanism of this reaction.

SUBMITTER: Pierdominici-Sottile G

PROVIDER: S-EPMC3033446 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Proton transfer facilitated by ligand binding. An energetic analysis of the catalytic mechanism of Trypanosoma cruzi trans-sialidase.

Pierdominici-Sottile Gustavo G Roitberg Adrian E AE

Biochemistry 20110111 5

Trans-sialidase is a crucial enzyme for the infection of Trypanosoma cruzi, the protozoa responsible for Chagas' disease in humans. This enzyme catalyzes the transfer of sialic acids from mammalian host cells to parasitic cell surfaces in order to mask the infection from the host's immune system. It represents a promising target for the development of therapeutics to treat the disease and has been subject of extensive structural studies. Elaborate experiments suggested formation of a long-lived ...[more]

PMID: 21162542

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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.

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Proton transfer facilitated by ligand binding. An energetic analysis of the catalytic mechanism of Trypanosoma cruzi trans-sialidase.

Publications

Proton transfer facilitated by ligand binding. An energetic analysis of the catalytic mechanism of Trypanosoma cruzi trans-sialidase.

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