Glucosidase-Catalyzed Hydrolysis of α-d-Glucopyranosyl Pyridinium Salts:  Kinetic Evidence for Nucleophilic Involvement at the Glucosidation Transition State

Kinetic isotope effects (KIEs) on the yeast α-glucosidase-catalyzed hydrolysis of two α-d-glucopyranosyl pyridinium salts were measured at 25 °C and pH = 6.8. The measured KIEs on kcat for the 2H-2, 13C-1, and 15N-1‘ labeled substrates α-d-glucopyranosyl pyridinium bromide (1) and α-d-glucopyranosyl isoquinolinium bromide (2) were, respectively, 1.115 ± 0.006 and 1.106 ± 0.009, 1.028 ± 0.006 and 1.027 ± 0.005, and 1.019 ± 0.007 and 0.985 ± 0.005. KIEs for the spontaneous hydrolysis of α-d-glucopyranosyl 4‘-bromoisoquinolinium bromide (3) were measured at 80 °C and pH = 6.8. The measured KIEs on khyd for the 2H-1, 2H-2, 13C-1, and 15N-1‘ labeled substrate α-d-glucopyranosyl 4‘-bromoisoquinolinium bromide (3) were, respectively, 1.189 ± 0.009, 1.094 ± 0.010, 1.005 ± 0.002, and 1.015 ± 0.004. The KIEs obtained in this study of yeast α-glucosidase-catalyzed hydrolysis reactions are consistent with a transition state which involves a small degree of nucleophilic attack by an enzyme active site carboxylate group at the anomeric carbon center, whereas the corresponding transition state for the uncatalyzed reaction in water does not involve any nucleophilic assistance. In addition, the equilibrium isotope effects for deprotonation of the conjugate acid (Ka14/Ka15) of isoquinoline was measured to be 1.0216 ± 0.0005.