The sialidase from Micromonospora viridifaciens (MvS) is inactivated by the sialic acid analogue 5-acetamido-2,3,5-trideoxy-3-fluoro-D-erythro-β-L-manno-non-2-ulopyranoson- yl fluoride (DiFSA), and by the Kdn analogue 2,3-dideoxy-3-fluoro-D-erythro-β-L-manno-non-2-ulopyranosonyl fluoride (DiFKdn). The second-order rate constant for inhibition of MvS by DiFSA at 25 C at a pH of 7.00 is 3.60 106 M–1 s–1. Whereas, the corresponding rate constant for inhibition by the Kdn inactivator is approximately one thousand-fold smaller (2.92 103 M–1 s–1 at 25 C and with a pH of 5.25). This decrease in activity on substitution of an acetamido group (DiFSA) for a hydroxyl functionality (DiFKdn) is remarkably similar to that for MvS-catalyzed hydrolysis of 4-nitrophenyl glycosides of sialic acid and Kdn. These observations are consistent with the difluoro containing inhibitors being 'mechanism-based' inactivators and that the hydrophobic pocket of MvS providing approximately 22 kJ/mol transition state stabilization by virtue of its hydrophobic interaction with the 5-acetamido group.
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Thesis advisor: Bennet, Andrew
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