STRUCTURAL FEATURES OF RED KIDNEY BEAN á-AMYLASE INHIBITOR IMPORTANT IN BINDING WITH á-AMYLASE

The structural features of the glycoprotein α-amylase inhibitor from red kidney bean (Phaseolus vulgaris) important for binding to α-amylase were examined. The inhibitor contained 13.0% carbohydrate. The carbohydrate portion of the inhibitor is composed of 25 mannose, 2 xylose, 1 fucose and 17 N-cetylglucosamine residues per mol. Seventy (70) percent of the neutral carbohydrates were removed from the inhibitor by treatment with endo-β- N -acetylglucosaminidase H. The carbohydrate remaining attached to the protein consisted of 7 mannose, 2 xylose, and 1 fucose residues (N-acetylogucosamine was not determined). The intact glyco chains obtained from the pronase digest of the inhibitor failed to inhibit β-amylase (3.3 times 10⁵ fold molar excess of glyco chains to enzyme) when preincubated with the enzyme at 30°C for 30 min at pH 6.9 before adding starch as substrate.
Oxidation of one tryptophan residue of the α-amylase inhibitor with N-bromosuccinimide at pH 6.0 led to a 50% loss in inhibitory activity. Periodate oxidation of α-amylase inhibitor led to less of two tyrosine and one methionine residues per mole of inhibitor within 15 min. There was not a direct correlation between modification of these residues and loss of inhibitory activity. Modification of three of the five histidine residues with diethylpyrocarbonate resulted in about 50% loss in inhibitory activity.