EFFECT OF SUBSTRATE AGLYCON ON ENZYME MECHANISM IN THE REACTION OF SIALIDASE FROM INFLUENZA-VIRUS

The effect of substrate aglycon on enzyme mechanism of sialidase from influenza virus was investigated by kinetic isotope effects using the substrates 4-methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid (Neu5 Ac alpha 2MU) and p-nitrophenyl-N-acetyl-alpha-D-neuraminic acid (Neu5 Ac alpha 2PNP). The kinetic isotope effect on V-max (V-beta D), at pH 6.0, as revealed by direct comparison of rates obtained with Neu5Ac al pha 2MU and the [3,3-H-2]-substituted substrate analogue, was shown to be inverse, This indicates that sialidase-catalysed hydrolysis of Neu 5Ac alpha 2MU 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 im inverse effec t on V-max at pH 6.0 was observed when using Neu5Ac alpha 2PNP and the [3,3-H-2]-substituted substrate. A mechanism by which hydrolysis proc eeds through an alpha-lactone intermediate has been proposed by Guo et al, [8]. We propose that the differences in V-beta D for the substrat es investigated are due primarily to the differing properties of the a glycon leaving groups, which may result in influenza virus sialidase c atalysing substrate hydrolysis by a similar mechanism with alternative , stabilisation of transition state.