Effect of neutral pyridine leaving groups on the mechanisms of influenza type a viral sialidase-catalyzed and spontaneous hydrolysis reactions of alpha-D-n-acetylneuraminides

A reagent panel, comprised of five pyridinium salts of alpha-D-N-acetylneur aminic acid, was synthesized and then used to probe enzymatic (alpha-sialid ase) and nonenzymatic mechanisms of neuraminide hydrolysis. Spontaneous hyd rolysis of the pyridinium salts proceeded via two independent pathways, whe re unassisted C-N bond cleavage was the rate-determining step. Cationic spe cies (i.e., anomeric carboxylate protonated) displayed apparent pK(a) value s 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 c alculated from the hydrolysis of pyridinium alpha-D-N-acetylneuraminide (De lta 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 no t assist in the departure of neutral pyridine leaving groups. Enzymatic hyd rolysis was studied using an influenza viral alpha-sialidase (A/Tokyo/3/67) which was recombinantly expressed using a baculovirus/insect cell expressi on system. Sialidase protein was purified by a combination of density gradi ent centrifugation and gel filtration chromatography. Kinetic parameters fo r the enzymatic hydrolysis of the pyridinium salts were measured at 37 degr ees C and at pH values of 6.0 and 9.5. The beta(1g) values derived for k(ca t)/K-m and k(cat) were essentially zero, indicating that chemical transform ations/events are not rate-determining. Rather, this observation is consist ent 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 Mi chaelis complex and k(cat) is determined by the virtual transition state ma de up of two separate conformational events.