Influenza A and B viruses contain two proteins on their surface: hemaglutinin (HA) and neuraminidase (NA) in addition to the M2 ion channel. HA binds to sialic acid containing receptors on the surface of host cells, facilitating the process of membrane fusion and allowing the virus to gain entry to the cell. In order for the influenza virus to efficiently break free from infected cells and to continue infecting new cells, sialic acid receptors must be destroyed. NA, which is a sialidase, catalyzes hydrolysis of terminally linked sialic acid and functions as the receptor destroying element of influenza A and B viruses. ..Two influenza M2 ion channel inhibitors Amantadine and Rimantadine have been approved for use; however they are extremely susceptible to drug resistant mutations and currently, the most commonly used drugs for seasonal and pandemic influenza function as NA competitive inhibitors. The NA targeted drugs, Tamiflu (oseltamivir phosphate) and Relenza (zanamivir), are approved and commonly used. The emergence of the 2009 H1N1 swine-origin influenza pandemic led to over 4 billion US dollars in combined sales of Tamiflu and Relenza (Roche and GSK earnings reports). Although there has been a great deal of clinical and pharmacological research done on the neuraminidase, there are still many questions relating to basic biochemical research that are left to be determined, especially regarding substrate binding and the mechanism of catalysis. Many structures of NA are available from many serotypes and they all contain very highly conserved active site residues, however, the precise mechanism of the influenza NA is still unknown and the binding of the original substrate, α2,3 or α2,6 linked sialic acid, is also uncharacterized. ..In this proposal, we set out to pinpoint the precise NA mechanism with direct crystallographic evidence in combination with biochemical assays and some NMR studies. Key active site residues will be substituted with alternate amino acids and soluble NA will be produced using a reliable baculovirus expression system. The effects of the substitutions on the enzymatic activity and active site structure/function will be analyzed in detail and provide insight into the design of novel influenza NA inhibitors. Some of the mutations/substitutions will result in NA inactivation, however this opens up the possibility to obtain a complex structure of NA with the original substrate, α2,3 or α2,6 linked sialic acid polysaccharide (LSTa or LSTc). This will allow for a highly detailed look at the influenza NA mechanism and substrate binding for the first time. We hypothesize that the NA 150-loop, which is responsible for forming the additional active site 150-cavity, is also involved in binding the original α2,3 or α2,6 substrate and may have a significant role in pairing with HA and pathogenicity.