title: Parameters involved in binding of porcine pancreatic alpha-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 alpha-amylase (MW 52,000) at pH 5.40. The single sulfhydryl group of the inhibitor and the two sulfhydryl groups of alpha-amylase are not involved in recognition and binding. Chloride ions, required for activity of alpha-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 alpha-amylase binds with the inhibitor. An increase in the ionic strength of the solvent increases the rate of binding of the inhibitor with alpha-amylase; a decrease in the dielectric constant decreases the rate of binding; and decreasing the temperature increases the dissociation constant, Kd, of the complex. These data support the hypothesis that hydrophobic interaction is of primary importance in complex formation. The activation energy, Ea, 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-alpha-D-maltoside, the Ea for complex formation was 4.1 kcal/mol at pH 6.90.
authors.affiliation: Departamento de Alimentos e Nutricão Experimental, Faculdade de Ciencias Farmacêuticas, Universidade de São Paulo, Brazil.
authors.forename: J R
authors.initials: JR
authors.lastname: Whitaker
authors.forename: F F
authors.initials: FF
authors.lastname: Filho
authors.forename: F M
authors.initials: FM
authors.lastname: Lajolo
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D010179:Pancreas / Q000201:enzymology*; D010946:Plants, Medicinal; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine; D013696:Temperature; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D002712:Chlorides; D013438:Sulfhydryl Compounds; D000516:alpha-Amylases; D002118:Calcium