Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus

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Abstract
The effect of substrate aglycon on enzyme mechanism of sialidase from influenza virus was investigated by kinetic isotope effects using the substrates acid (Neu5Acα2MU) and acid (Neu5Acα2PNP). The kinetic isotope effect on , at pH 6.0, as revealed by direct comparison of rates obtained with Neu5Acα2MU and the [3,3‐2H]‐substituted substrate analogue, was shown to be inverse. This indicates that sialidase‐catalysed hydrolysis of Neu5Acα2MU proceeds with substantial positive charge development at the reaction centre in the transition state for the formation of the glycosyl cation‐enzyme intermediate. However, no such inverse effect on V max at pH 6.0 was observed when using Neu5Acα2PNP and the [3,32H]‐substituted substrate. A mechanism by which hydrolysis proceeds through an α‐lactone intermediate has been proposed by Guo et al. [8]. We propose that the differences in βD V for the substrates investigated are due primarily to the differing properties of the aglycon leaving groups, which may result in influenza virus sialidase catalysing substrate hydrolysis by a similar mechanism with alternative stabilisation of transition state.