300595-03-3Relevant articles and documents
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
Chou, Doug T. H.,Watson, Jacqueline N.,Scholte, Andrew A.,Borgford, Thor J.,Bennet, Andrew J.
, p. 8357 - 8364 (2000)
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 pK(a) values in the range of 0.4-0.7. However, spontaneous hydrolyses of the cationic and zwitterionic species had similar β(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 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 β(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 α-sialidase-catalyzed hydrolyses (Guo, X.; Laver, W. G.; Vimr, E.; Sinnott, M. L., J. Am. Chem. Soc. 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.
