Transition State Analysis of Vibrio cholerae Sialidase-Catalyzed Hydrolyses of Natural Substrate Analogues

A series of isotopically labeled natural substrate analogues (phenyl 5-N-acetyl-alpha-u-neuraminyl-(2 -> 3)-alpha-D-galactopyranosyl-(2 -> 4)-1-thio-beta-D-glucopyranoside; Neu5Ac alpha 2,3Lac beta SPh, and the corresponding 2 -> 6 isomer) were prepared chemoenzymatically in order to characterize, by use of multiple kinetic isotope effect (KIE) measurements, the glycosylation transition states for Vibrio cholerae sialidase-catalyzed hydrolysis reactions. The derived KIEs for Neu5Ac alpha 2,3Lac beta SPh for the ring oxygen (V-18/K), leaving group oxygen (V-18/K), C3-S deuterium (V-D/K-S) and C3-R deuterium (V-D/K-R) are 1.029 +/- 0.002, 0.983 +/- 0.001, 1.034 +/- 0.002, and 1.043 +/- 0.002, respectively. In addition, the KIEs for Neu5Ac alpha 2,6 beta SPh for C3-S deuterium (V-D/K-S) and C3-R deuterium (V-D/K-R) are 1.021 +/- 0.001 and 1.049 +/- 0.001, respectively. The glycosylation transition state structures for both Neu5Ac alpha 2,3Lac beta SPh and Neu5Ac alpha 2,6Lac beta SPh were modeled computationally using the experimental KIE values as goodness of fit criteria. Both transition states are late with largely cleaved glycosidic bonds coupled to pyranosyl ring flattening (H-4(5) half-chair conformation) with little or no nucleophilic involvement of the enzymatic tyrosine residue. Notably, the transition state for the catalyzed hydrolysis of Neu5Ac alpha 2,6 beta SPh appears to incorporate a lesser degree of general-acid catalysis, relative to the 2,3-isomer.