Brønsted analysis of an enzyme-catalyzed pseudo-deglycosylation reaction: mechanism of desialylation in sialidases.

The Micromonospora viridifaciens Y370G inverting mutant sialidase has been found to possess β-sialidase activity with various fluoro-substituted phenyl β-sialosides. A reagent panel of seven mono- and difluorophenyl β-d-sialosides was synthesized, and these compounds were used, in conjunction with the parent phenyl β-d-sialoside, to probe the mechanism of M. viridifaciens Y370G mutant sialidase-catalyzed hydrolyses. These hydrolysis reactions mimic the deglycosylation reaction step of the crucial tyrosinyl enzyme-bound intermediate that is formed during the corresponding wild-type sialidase reactions. The derived Bronsted parameter (βlg) on kcat/Km is −0.46 ± 0.02 for the four substrates that display significant activity, and these span a range of leaving group abilities (as judged by the pKa of their conjugate acids being between 7.09 and 9.87). The 4-fluoro, 2,3- and 2,5-difluorosubstrates display a diminished activity, whereas the 3,5-difluoro compound undergoes catalyzed hydrolysis exceedingly slowly....