title: Brønsted analysis of an enzyme-catalyzed pseudo-deglycosylation reaction: mechanism of desialylation in sialidases.
abstract: 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 beta-d-sialosides was synthesized, and these compounds were used, in conjunction with the parent phenyl beta-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 (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 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(H(2))(O)/k(D(2))(O)) 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 (IC(50) > 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.
authors.affiliation: Departments of Chemistry and Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada.
authors.forename: Fahimeh S
authors.initials: FS
authors.lastname: Shidmoossavee
authors.forename: Lydia
authors.initials: L
authors.lastname: Cheng
authors.forename: Jacqueline N
authors.initials: JN
authors.lastname: Watson
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bennet
mesh_terms: D002384:Catalysis; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008847:Micromonospora / Q000201:enzymology*; D020125:Mutation, Missense; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid