A reagent panel, comprised of five pyridinium salts of alpha-D-N-acetylneuraminic acid, was synthesized and then used to probe enzymatic (alpha-sialidase) and nonenzymatic mechanisms of neuraminide hydrolysis. Spontaneous hydrolysis of the pyridinium salts proceeded via two independent pathways, where unassisted C-N bond cleavage was the rate-determining step. Cationic species (i.e., anomeric carboxylate protonated) displayed apparent pK(a) values in the range of 0.4-0.7. However, spontaneous hydrolyses of the cationic and zwitterionic species had similar beta(1g) values of -1.22 +/-0.16 and -1.22 +/- 0.07, respectively. The results, plus the activation parameters calculated from the hydrolysis of pyridinium alpha-D-N-acetylneuraminide (Delta H double dagger = 112 +/- 2 kJ mol(-1) and Delta S double dagger = 28 +/- 4 J mol(-1) K-1), strongly suggest that the anomeric carboxylate does not assist in the departure of neutral pyridine leaving groups. Enzymatic hydrolysis was studied using an influenza viral alpha-sialidase (A/Tokyo/3/67) which was recombinantly expressed using a baculovirus/insect cell expression system. Sialidase protein was purified by a combination of density gradient centrifugation and gel filtration chromatography. Kinetic parameters for the enzymatic hydrolysis of the pyridinium salts were measured at 37 degrees C and at pH values of 6.0 and 9.5. The beta(1g) values derived for k(cat)/K-m and k(cat) were essentially zero, indicating that chemical transformations/events are not rate-determining. Rather, this observation is consistent with a model for alpha-sialidase-catalyzed hydrolyses (Cuo, X.; Laver, W. G.; Vimr, E.; Sinnott, M. L. J. Am. Chem. Sec. 1994, 116, 5572) in which k(cat)/K-m is determined by a conformational change of the first-formed Michaelis complex and k(cat) is determined by the virtual transition state made up of two separate conformational events.