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 beta-sialidase activity with various fluoro-substituted phenyl beta-sialosides. A reagent panel of seven mono- and difluorophenyl,8-D-sialosides was synthesized, and these compounds were used, in conjunction with the parent phenyl beta-o-sialoside, to probe the mechanism of M. viridrfaciens 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 (beta(lg)) on k(cat)/K-m 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. These observations, taken with solvent deuterium kinetic isotope effects (ki.i.,0//cD20) on the catalyzed hydrolysis of the 2-fluorophenyl substrate of 0.88 +/- 0.24 (k(cat)/K-m) and 1.16 0.12 (k(cat)) and the poor inhibition shown by phenol (IC50 > 1 mM), are consistent with glycosidic C-O cleavage being rate determining for both k(cat)/K-m and k(cat), with little or no protonation of the departing aryloxide leaving group. The kinetic data reported herein are consistent with rate-limiting glycoside hydrolysis occurring via two distinct transition states that incorporates a nonproductive binding component for the tighter binding substrates.