Identity and Properties of the Chloride Effector Binding Site in Hog Pancreatic α-Amylase

The Cl^- activated-amylase from mammalian sources has been shown previously to possess one Cl^- binding site per molecule (Levitzki, A., and Steer, M. L. (1974), Eur. J. Biochem. 41, 171). Upon binding of the Cl^- effector the ¿cat of the amylolytic reaction is increased 30-fold whereas the affinity toward the substrate remains unchanged. In the study presented here we have identified the Cl^- binding site as a single e-amino group of lysine. The pK of the unique amino group was found to be 9.1, significantly lower than the pH of a free e-amino group of lysine. This e-NH₂ group can be blocked by a 2,4-dinitrophenyl group upon treating the enzyme with 2,4-dinitrofluorobenzene at pH 7.9. The dinitrophenylamylase is devoid of Cl^- binding capacity but retains its substrate binding capacity. The dinitrophenylamylase also possesses the basal amylolytic activity characteristic of the unmodified Cl^- free enzyme, indicating that the catalytic machinery of the enzyme is not affected by dinitrophenylation. «-Limit dextrins and maltose which bind to the active site protect the enzyme against dinitrophenylation at least as effectively as the Cl^- effector. These observations indicate that the Cl^- binding lysyl residue is close to the active site and, upon binding, the Cl^- effector induces an enhancement in the catalytic efficiency.