Brønsted Analysis of an Enzyme-Catalyzed Pseudo-Deglycosylation Reaction: Mechanism of Desialylation in Sialidases

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• 作者：Fahimeh S. Shidmoossavee ; Lydia Cheng ; Jacqueline N. Watson ; Andrew J. Bennet
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：August 3, 2010
• 年：2010
• 卷：49
• 期：30
• 页码：6473-6484
• 全文大小：993K
• 年卷期：v.49,no.30(August 3, 2010)
• ISSN：1520-4995

文摘

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-<span class="smallcaps">dspan>-sialosides was synthesized, and these compounds were used, in conjunction with the parent phenyl β-<span class="smallcaps">dspan>-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<sub>lgsub>) on k<sub>catsub>/K<sub>msub> 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<sub>asub> 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<sub>H<sub>2sub>sub><sub>Osub>/k<sub>D<sub>2sub>sub><sub>Osub>) on the catalyzed hydrolysis of the 2-fluorophenyl substrate of 0.88 ± 0.24 (k<sub>catsub>/K<sub>msub>) and 1.16 ± 0.12 (k<sub>catsub>) and the poor inhibition shown by phenol (IC<sub>50sub> > 1 mM), are consistent with glycosidic C−O cleavage being rate determining for both k<sub>catsub>/K<sub>msub> and k<sub>catsub> 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.