Combination of black bean (Phaseolus vulgaris) amylase inhibitor with modified pancreatic alpha-amylase

ABSTRACT Several amino acid residues important for the action of porcine pancreatic α‐amylase on starch were modified using specific reagents: histidine groups by photooxidation with rose bengal or by diethylpyrocarbonate; cysteine by dithiobis nitrobenzoic acid; tryptophan by N‐bromosuccimide and tyrosine by hydroxyl acetylation with N‐acetylimidazole. These modifications, with the exception of cysteine, reduced the amylase activity but none of them alone was able to alter significantly the inhibition of the enzyme by a purified black bean (Phaseolus vulgaris) amylase inhibitor. Only by photooxidation that can modify several groups at the same time was the inhibitor action eliminated. The results suggested that the histidine of the amylase active site and tyrosine of the substrate binding site are not important for binding to the bean inhibitor. The terminal sugars of the amylase inhibitor were identified as mannose and xylose. Periodate oxidation of the carbohydrate moiety caused total loss of activity. The treatment of the inhibitor with α‐mannosidase did not alter its inhibitor action on α‐amylase.