Site-directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design

The neuraminidase of influenza virus is a surface glycoprotein that catalyz es the hydrolysis of glycosidic linkages between terminal sialic acids and adjacent sugar moieties. Neuraminidase function is critical for the spread of virus to new cells, and if the enzyme activity is inhibited, then virus infection is abrogated. The neuraminidase active site is conserved in all i nfluenza type-A and type-B isolates, which makes it an excellent target for drug design. To determine the potential for resistance to develop against neuraminidase inhibitors, we have constructed mutations in seven of the con served active-site residues of a type B (B/Lee/40) neuraminidase and analyz ed the effect of the altered side chains on enzyme activity. There is a red uction in k(cat) in all our mutants. A transition-state analogue inhibitor shows variation in K-i with the mutant neuraminidases, allowing interpretat ion of the effects of mutation in terms of transition-state binding and pro duct release. The results show that Tyr409 is the most critical residue for enzyme activity, but that Asp149, Arg223, Glu275 and Arg374 also play impo rtant roles in enzyme catalysis. Based on the pH profile of neuraminidase a ctivity of the D149E mutant protein, we conclude that Asp149 is not a proto n donor, but is involved in stabilizing the transition state. If designed i nhibitors are targeted to these residues where mutations are highly deleter ious, particularly Tyr409, Glu275 and Asp149, the virus is unlikely to gene rate resistance to the drug.