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

Abstract

The Cl- activated .alpha.-amylase from mammalian sources had been shown previously to possess 1 Cl- binding site per molecule. Upon binding of the Cl- effector the kcat of the amylolytic reaction is increased 30-fold; the affinity toward the substrate remains unchanged. The Cl- binding site was identified as a single .epsilon.-amino group of lysine. The pK of the unique amino group was 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 least as effectively as the Cl- effector. The Cl- binding lysyl residue may be close to the active site, and upon binding the Cl- effector induces an enhancement in the catalytic efficiency.