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Glycobiology vol 7 no 8 pp. 1237-1246, 1997
© 1997
Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase
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Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP Rua Botucatn 862, 8° andar, 04023-062, São Paulo, S.P. Brazil
1 Laboratório de Xenodiagnóstico-Instituto Dante Pazzanese de Cardiologia São Paulo, Brazil
2 Department of Medical and Molecular Parasitology, New York University Medical Center New York, NY, 10010 USA
3To whom correspondence should be addressed at Disciplina de Biologia Celular, R. Botucatu 862 8* 04023-062 Sãb Paulo, S.P. Brazil
Received on April 28, 1997; revised on July 2, 1997; accepted on July 2, 1997
Trypanosoma cruzi, the agent of Chagas disease, expresses on its surface a trans-sialidase that catalyzes preferentially the transference of 
-2,3-linked sialic acid to acceptors containing terminal β-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 β-galactosyl residues have a unique behavior with respect to the reaction temperature. While the hydrolysis rate of sialyUactose increases continuously up to 35°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°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 Km for the acceptor from 13°C to 33°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 β-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.
Trypanosoma cruzi sialidase kinetics reaction mechanism temperature
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