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

• Published in: FEBS letters Vol. 372; no. 2; pp. 148 - 150
• Main Authors: Tiralongo, J., Pegg, M.S., von Itzstein, M.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 25.09.1995
• Subjects: [formula omitted] acid; acid; Enzyme Activation; Hymecromone - analogs & derivatives; Hymecromone - metabolism; influenza A virus; Influenza virus; Kinetic isotope effect; Neuraminidase - metabolism; Orthomyxoviridae - enzymology; Sialidase; Substrate aglycon; Substrate Specificity; Transition state; 4- Methylumbelliferyl-N- acetyl-α- d-neuraminic acid; Kinetic isotope effect; Influenza virus; Substrate aglycon; Transition state; p- Nitrophenyl-N- acetyl-α- d-neuraminic acid; Sialidase
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(95)00967-E

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-α- d-neuraminic acid (Neu5Acα2MU) and p- nitrophenyl-N- acetyl-α- d-neuraminic acid (Neu5Acα2PNP). The kinetic isotope effect on V max ( β D V) , 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 [3,3 2H]-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.