Identification of essential ionizable groups in active site of Aspergillus niger alpha-glucosidase

A kinetic study was done to identify the ionizable groups in the active site of Aspergillus niger alpha-glucosidase (ANGase). From dependence of V and K-m values on pH, we obtained the ionization constants of essential ionizable groups 1 and 2 of free enzyme; pKe(1) = 3.2 and pKe(2) = 6.4. When the dielectric constant of the reaction mixture was decreased, the pKe(1) and pKe(2) were shifted to higher values, The ionization heats (Delta H's) of ionizable groups 1 and 2 were measured to be - 0.4 kcal/mol and 0 kcal/mol, respectively, The water-soluble carbodiimide (WSC), a specific reagent for carboxyl groups, inactivated the enzyme activity completely, and maltose as substrate decreased the inactivation, The WSC did not modify the free Cys, These findings suggest that the essential ionizable groups of ANGase are two kinds of carboxyl groups: one is a charged type (-COO-, ionizable group 1), and the other is a protonated type (-COOH, ionizable group 2).