Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Autor Ribeirao, Marcelo Autor UNIFESP Google Scholar
Pereira-Chioccola, Vera Lucia Autor UNIFESP Google Scholar
|Eichinger, D. Google Scholar
Rodrigues, Mauricio Martins Autor UNIFESP Google Scholar
Schenkman, Sergio Autor UNIFESP Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
INST DANTE PAZZANESE CARDIOL
NYU
Resumo Trypanosoma cruzi, the agent of Chagas disease, expresses on its surface a trans-sialidase that catalyzes preferentially the transference of alpha-2,3-linked sialic acid to accepters containing terminal beta-galactosyl residues, instead of the typical hydrolysis reaction, found in most sialidases. the trans-sialidase is responsible for the acquisition of the host sialic acid by this protozoan parasite, which does not synthesize sialic acids, Here, we have studied some kinetic properties of a recombinant trans-sialidase expressed in Escherichia coli, We found that it has sequential-type kinetics for the transferase reaction, as shown for the parasite-derived enzyme, the rates of sialic acid transfer to water (hydrolysis), and to beta-galactosyl residues have a unique behavior with respect to the reaction temperature, While the hydrolysis rate of sialyllactose increases continuously up to 35 degrees C, the temperature for the maximal rate of trans-glycosylation depends on the acceptor concentration, At low acceptor concentrations the rate of trans-glycosylation is maximal at 13 degrees C and independent of the amount of sialic acid donors, With increasing acceptor concentrations, maximal rates of trans-glycosylation are shifted to higher temperatures, This finding is explained by an 8-fold increase in the K-m for the acceptor from 13 degrees C to 33 degrees C, Differences in hydrolysis and transfer rates were also obtained by using 4-methyl-umbelliferyl-N-acetyl-neuraminic acid. However, its hydrolysis rate is much higher than the rate of transference to lactose, suggesting that a long-lived enzyme-sialosyl intermediate is not formed, in addition, lactose does not increase the rate of methyl-umbelliferone release at any temperature, indicating that the rate limiting step is the aglycon release, Based on these results we propose that trans-glycosylation in T.cruzi sialidase is favored by the existence of a binding site for beta-galactosyl residues, which accepts the new glycosidic bond as sialic acid is released from the donor, With increasing temperature the affinity for the acceptor decreases, resulting in a concomitant increase in the rate of transfer to water, which, in turn, can be suppressed by increasing the acceptor concentration.
Palavra-chave Trypanosoma cruzi
sialidase
kinetics
reaction mechanism
temperature
Idioma Inglês
Data de publicação 1997-12-01
Publicado em Glycobiology. Oxford: Oxford Univ Press, v. 7, n. 8, p. 1237-1246, 1997.
ISSN 0959-6658 (Sherpa/Romeo, fator de impacto)
Publicador Oxford Univ Press
Extensão 1237-1246
Fonte http://dx.doi.org/10.1093/glycob/7.8.1237
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:A1997YK76700022
Endereço permanente http://repositorio.unifesp.br/handle/11600/25815

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