Identification of Essential Ionizable Groups in Active Site of Aspergillus niger αα-Glucosidase

概要

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A kinetic study was done to identify the ionizable groups in the active site of Aspergillus niger α-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 (ΔH'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).
社団法人日本農芸化学会の論文
1997-03-23