Effect of Neutral Pyridine Leaving Groups on the Mechanisms of Influenza Type A Viral Sialidase-Catalyzed and Spontaneous Hydrolysis Reactions of α-d-N-Acetylneuraminides

Abstract

A reagent panel, comprised of five pyridinium salts of α-d-N-acetylneuraminic acid, was synthesized and then used to probe enzymatic (α-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 pKa values in the range of 0.4−0.7. However, spontaneous hydrolyses of the cationic and zwitterionic species had similar βlg values of −1.22 ± 0.16 and −1.22 ± 0.07, respectively. The results, plus the activation parameters calculated from the hydrolysis of pyridinium α-d-N-acetylneuraminide (ΔH⧧ = 112 ± 2 kJ mol-1 and ΔS⧧ = 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 α-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 °C and at pH values of 6.0 and 9.5. The βlg values derived for kcat/Km and kcat were essentially zero, indicating that chemical transformations/events are not rate-determining. Rather, this observation is consistent with a model for α-sialidase-catalyzed hydrolyses (Guo, X.; Laver, W. G.; Vimr, E.; Sinnott, M. L. J. Am. Chem. Soc. 1994, 116, 5572) in which kcat/Km is determined by a conformational change of the first-formed Michaelis complex and kcat is determined by the virtual transition state made up of two separate conformational events.