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

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.