Is There a Covalent Intermediate in the Viral Neuraminidase Reaction? A Hybrid Potential Free-Energy Study

The neuraminidase from the influenza virus is essential for maintaining viral infectivity as it aids in the transmission of the virus between cells. Although there are large variations in the amino acid sequences of neuraminidases from different influenza strains, there are several amino acids in the active site region of the protein that are strictly conserved. This has raised hopes that a single neuraminidase inhibitor and, hence a drug, can be found that is effective against all influenza strains. In this paper, we examine with theoretical simulation techniques one aspect of the reaction catalyzed by the viral neuraminidase that could be important for inhibitor design studiesnamely, whether a covalently bound complex can be formed between the enzyme and the sialosyl cation intermediate that occurs during the reaction. We used a hybrid semiempirical quantum mechanical/molecular mechanical (QM/MM) potential in conjunction with potential of mean force calculations to determine the free-energy profiles for formation of the covalent intermediate and its hydrolysis to sialic acid and for the direct hydroxylation of the sialosyl cation to sialic acid. Ab initio QM calculations were used to check the validity of the semiempirical results. We find that direct hydroxylation of the sialosyl cation is the energetically preferred pathway but not by so much that the possibility of being able to design covalently bound inhibitors need be completely abandoned.