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

Eur J Biochem. 1992 Jul 1;207(1):335-43. doi: 10.1111/j.1432-1033.1992.tb17055.x.


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-alpha-D-neuraminic acid and the [3,3-2H]-substituted substrate revealed beta-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, beta DV/Km was slightly increased by 2% and 5% at pD 6.0 and 9.5 respectively, while beta DV was unchanged. Solvent isotope effects of 1.74 were obtained for both beta DV/Km and beta DV at pD 9.5, with beta DV/Km decreasing and beta DV remaining constant at acidic pD. 1H-NMR experiments confirmed that the initial product of the reaction is the alpha-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Carbohydrate Conformation
  • Cations
  • Deuterium
  • Hydrogen-Ion Concentration
  • Influenza A virus / enzymology*
  • Isotope Labeling
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Neuraminidase / metabolism*
  • Sialic Acids / metabolism
  • Substrate Specificity


  • Cations
  • Sialic Acids
  • Deuterium
  • Neuraminidase