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 degrees C, the derived kinetic parameters are k(inact)/K-i = (3.9 +/- 0.8) x 10(6) (mol/L)(-1) s(-1) and K-i = 1.7 +/- 0.4 mu mol/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 alpha-D-sialoside, k(cat)/K-m = (7.2 +/- 2.8) x 10(6) (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.