Inhibitory efficiencies for mechanism-based inactivators of sialidases

Here we describe the measurement of the inactivation rate constants for the mechanism-based inactivator 2,3-difluorosialic acid acting upon the sialidase from Micromonospora viridifaciens. Using double mixing stopped-flow experiments conducted in a 3-(N-morpholino)propanesulfonic acid buffer (100 mmol/L, pH 7.00) at 25 °C, the derived kinetic parameters are kinact/Ki = (3.9 ± 0.8) × 106 (mol/L)–1 s–1 and Ki = 1.7 ± 0.4 μmol/L. We demonstrate that the inhibitory efficiency of the inactivation event is similar to the catalytic efficiency for this sialidase acting upon a typical substrate, 4-methylumbelliferone α-d-sialoside, kcat/Km = (7.2 ± 2.8) × 106 (mol/L)–1 s–1. Furthermore, we show that the catalytic efficiencies for inactivation and hydrolysis by the Kdnase from Aspergillus fumigatus are similar for the corresponding Kdn-analogues. We conclude that the deactivating effect of incorporating an axial 3-fluoro substituent onto the sialic acid scaffold is comparable to the enhanced activation that occurs when the 4-methylumbelliferone leaving group is changed to the more nucleofugal fluoride ion.