Characterisation of an ionisable group involved in binding and catalysis by sialidase from influenza virus.
The effect of pH on the kinetics of sialidase purified from influenza virus (A/Tokyo/3/67, H2N2) was investigated. A pK of 9.0 for inhibition of the enzyme by three competitive inhibitors, due to an ionisable group in the active site, was observed. A similar pK was observed for V/Km for the fluorogenic substrate 2-(4-methylumbelliferyl)-N-acetyl-alpha-D-neuraminic acid. However, the shape of the V/Km profile indicates that this substrate is sticky. Solvent perturbation experiments indicated that the observed ionisable active site group is likely to be a cationic amino acid. The results provide evidence against the hypothesis that Glu 276 acts as a proton donor in the enzyme reaction and supports the proposal of a role for one of the active site cationic amino acids in binding and catalysis.
Chong
A K
AK
School of Pharmaceutical Chemistry, Victorian College of Pharmacy Ltd., Parkville, Australia.
Pegg
M S
MS
von Itzstein
M
M
D001665:Binding Sites; D001667:Binding, Competitive; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives; D009980:Influenza A virus / Q000201:enzymology*; D007700:Kinetics; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005456:Fluorescent Dyes; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009439:Neuraminidase