Evidence for a sialosyl cation transition‐state complex in the reaction of sialidase from influenza virus

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Abstract
The enzyme mechanism of sialidase from influenza virus has been investigated by kinetic isotope methods, NMR, and a molecular dynamics simulation of the enzyme‐substrate complex. Comparison of the reaction rates obtained with the synthetic substrate 4‐methylumbelliferyl‐N‐acetyl‐α‐D‐neuraminic acid and the [3,3‐2H]‐substituted substrate revealed β‐deuterium isotope effects for V/Km ranging over 1.09–1.15 in the pH range 6.0–9.5, whereas the effects observed for V in this pH range increased from 0.979 to 1.07. In D2O, βDV/Km was slightly increased by 2% and 5% at pD 6.0 and 9.5 respectively, while βDV was unchanged. Solvent isotope effects of 1.74 were obtained for both βDV/Km and βDV at pD 9.5, with βDV/Km decreasing and βDV remaining constant at acidic pD. 1H‐NMR experiments confirmed that the initial product of the reaction is the α‐anomer of N‐acetyl‐D‐neuraminic acid. Molecular dynamics studies identified a water molecule in the crystal structure of the sialidase‐N‐acetyl‐D‐neuraminic acid complex which is hydrogen‐bonded to Asp151 and is available to act as a proton donor source in the enzyme reaction. The results of this study lead us to propose a mechanism for the solvent‐mediated hydrolysis of substrate by sialidase that requires the formation of an endocyclic sialosyl cation transition‐state intermediate.

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