Mutation in the influenza virus neuraminidase gene resulting in decreased sensitivity to the neuraminidase inhibitor 4-guanidino-Neu5Ac2en leads to instability of the enzyme.
We previously isolated a variant of the influenza virus NWS/G70C, with a decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en in vitro, which has a mutation in one of the conserved residues of the neuraminidase Glu 119 to Gly. Despite the mutation, purified neuraminidase demonstrated the same specific activity as the parent neuraminidase. In contrast, characterization of a similar mutant by another group revealed a low specific activity of the enzyme. We confirm here that the specific activity of our variant is the same as that of the parent, but report that this mutation makes the enzyme inherently unstable, at high and low temperatures, either on the virion or as purified neuraminidase. Thus, for a valid determination of specific activity the concentration of native NA needs to be determined at the time of enzyme assay. Structurally, the instability may be partially explained by the introduction of a side chain (Gly), which carries a greater entropy penalty in condensation of the structure from the unfolded to the folded state and this, together with the loss of stabilizing interaction between Glu 119 and its neighbors in the active site, is not compensated for by the water molecule occupying the position of the carboxylate group (6).
McKimm-Breschkin
J L
JL
Biomolecular Research Institute, Parkville, Victoria, Australia. jennyb@mel.dbe.csiro.au
McDonald
M
M
Blick
T J
TJ
Colman
P M
PM
D000818:Animals; D002460:Cell Line; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005814:Genes, Viral / Q000235:genetics; D006146:Guanidines; D006801:Humans; D009154:Mutation; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009975:Orthomyxoviridae / Q000201:enzymology* / Q000235:genetics; D011714:Pyrans; D012794:Sialic Acids / Q000494:pharmacology*; D013696:Temperature; D053243:Zanamivir
D004791:Enzyme Inhibitors; D006146:Guanidines; D011714:Pyrans; D012794:Sialic Acids; D009439:Neuraminidase; D053243:Zanamivir