title: Identity and properties of the chloride effector binding site in hog pancreatic alpha-amylase.
abstract: 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.
mesh_terms: D000681:Amylases / Q000378:metabolism*; D000818:Animals; D001051:Apoenzymes; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology*; D004140:Dinitrophenols / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology*; D011485:Protein Binding; D013552:Swine
chemical_list: D001051:Apoenzymes; D002712:Chlorides; D004140:Dinitrophenols; D004492:Edetic Acid; D000681:Amylases; D002118:Calcium