Glucosidase-catalyzed hydrolysis of alpha-D-glucopyranosyl pyridinium salts: Kinetic evidence for nucleophilic involvement at the glucosidation transition state

Kinetic isotope effects (KIEs) on the yeast alpha-glucosidase-catalyzed hydrolysis of two alpha-D-glucopyranosyl pyridinium salts were measured at 25 degrees C and pH = 6.8. The measured KIEs on k(cat) for the H-2-2, C-13-1, and N-15-1' labeled substrates alpha-D-glucopyranosyl pyridinium bromide (1) and alpha-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 alpha-D-glucopyranosyl 4'-bromoisoquinolinium bromide (3) were measured at 80 degrees C and pH = 6.8, The measured KIEs on k(hyd) for the H-2-1, H-2-2, C-13-1, and N-15-1' labeled substrate alpha-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 alpha-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 (K-a(14)/K-a(15)) of isoquinoline was measured to be 1.0216 +/- 0.0005.