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 Brønsted parameter (β_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 pK_a 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 (k_H2O/k_D2O) 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.