Identity and properties of the chloride effector binding site in hog pancreatic alpha-amylase.

The Cl- activated alpha-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 kcat 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 epsilon-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 epsilon-amino group of lysine. This epsilon-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. alpha-Limit dextrins and maltose which bind to the active site protect the enzyme against dinitrophenylation at lea-st 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.