Parameters involved in binding of porcine pancreatic α-amylase with black bean inhibitor: role of sulfhydryl groups, chloride, calcium, solvent composition and temperature

Abstract

The amylase inhibitor of black (kidney) beans ( Phaseolus vulgaris; MW 53 000) forms a 1:1 stoichiometric complex with porcine pancreatic α-amylase (MW 52 000) at pH 5.40. The single sulfhydryl group of the inhibitor and the two sulfhydryl groups of α-amylase are not involved in recognition and binding. Chloride ions, required for activity of α-amylase at both pH 5.40 and 6.90, are important for inhibitor-enzyme binding at pH 6.90 but not at pH 5.40. Calcium-free α-amylase binds with the inhibitor. An increase in the ionic strength of the solvent increases the rate of binding of the inhibitor with α-amylase; a decrease in the dielectric constant decreases the rate of binding; and decreasing the temperature increases the dissociation constant, K d, of the complex. These data support the hypothesis that hydrophobic interaction is of primary importance in complex formation. The activation energy, E a, for complex formation was found to be 12.4 kcal/mol at pH 5.40 and 24.2 kcal/mol at pH 6.90. In the presence of the poor substrate, p- nitrophenyl-α- D-maltoside , the E a for complex formation was 4.1 kcal/mol at pH 6.90.