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

• Published in: Biochimie Vol. 70; no. 9; pp. 1153 - 1161
• Main Authors: Whitaker, J.R., Finardi Filho, F., Lajolo, F.M.
• Format: Journal Article
• Language: English
• Published: Paris Elsevier Masson SAS 01.09.1988; Elsevier
• Subjects: alpha-Amylases - antagonists & inhibitors; alpha-Amylases - metabolism; AMILASAS; AMYLASE; AMYLASES; Analytical, structural and metabolic biochemistry; Animals; BIOCHEMISTRY; BIOCHIMIE; Biological and medical sciences; BIOQUIMICA; black bean inhibitor; Calcium - metabolism; Chlorides - metabolism; ENZYME INHIBITORS; Enzymes and enzyme inhibitors; Fabaceae - enzymology; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolases; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; PANCREAS; Pancreas - enzymology; PHASEOLUS VULGARIS; Plants, Medicinal; porcine pancreatic α-amylase; Sulfhydryl Compounds - metabolism; sulfhydryl groups; Swine; Temperature; black bean inhibitor; sulfhydryl groups; porcine pancreatic α-amylase; Temperature; Calcium; Hydrolase; Enzyme; Thiohydroxyl group; Enzyme inhibitor; Molecular interaction; Pig; Vertebrata; Mammalia; Leguminosae; Phaseolus vulgaris; Dicotyledones; Chlorides; Angiospermae; Spermatophyta; Artiodactyla; α-Amylase; Pancreas; Ungulata
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/0300-9084(88)90180-0

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.