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 vir al infectivity as it aids in the transmission of the virus between cells. A lthough there are large variations in the amino acid sequences of neuramini dases from different influenza strains, there are several amino acids in th e active site region of the protein that are strictly conserved. This has r aised hopes that a single neuraminidase inhibitor and, hence a drug, can be found that is effective against all influenza strains. In this paper, we e xamine with theoretical simulation techniques one aspect of the reaction ca talyzed by the viral neuraminidase that could be important for inhibitor de sign studies-namely, whether a covalently bound complex can be formed betwe en the enzyme and the sialosyl cation intermediate that occurs during the r eaction. We used a hybrid semiempirical quantum mechanical/molecular mechan ical (QM/MM) potential in conjunction with potential of mean force calculat ions to determine the free-energy profiles for formation of the covalent in termediate and its hydrolysis to sialic acid and for the direct hydroxylati on of the sialosyl cation to sialic acid. Ab initio QM calculations were us ed 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.