Structure and Mechanism of Action of an Inverting Mutant Sialidase.

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682−12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3‘-sialyllactose, the derived Bronsted parameters (βlg) on kcat and kcat/Km are −0.63 ± 0.05 and −0.80 ± 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C−O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium α-d-N-acetylneuraminide (DHP-αNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via SN1 and SN2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Struc...