Abstract
The essential ionizable groups of rice α-glucosidase II were identified by kinetics and chemical modification methods. The pKe of ionizable group 1 (on the acidic side) was 3.1, and that of ionizable group 2 (on the alkaline side) was 6.1. The heat of ionization of ionizable group 2, ΔH2, was -1.7 kcal/mol. The pH of both the pKe1 and pKe2 values increased by about 0.5 when the dielectric constant of the reaction medium was lowered by adding 20% methanol. The chemical modification of the active site in rice α-glucosidase II with conduritol B epoxide, an affinity labeling reagent, resulted in inactivation of the enzyme. The inactivation followed pseudo-first-order kinetics. The enzyme was protected from inactivation by a competitive inhibitor, Tris, and the partially inactivated enzymes showed only a decrease in V values and no change in Km. These results strongly suggest that the essential ionizable groups of rice α-glucosidase II are -C00-and -COOH.
