Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans

Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivale nt inhibitors with the subunit stoichiometry of (alpha beta)(2) complex, ha ve been inferred to contain unique arginine, tryptophan, and tyrosine resid ues essential for the inhibitory activity. To test the validity of this inf erence, an attempt was made to identify the essential amino acid residues o f a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by usin g the chemical modification technique combined with amino acid sequencing a nd mass spectrometry. Exhaustive modification of the arginine residues by p henylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bro mosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in cr-amy lase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-ac etylimidazole or by tetranitromethane, resulting in a concomitant loss of m ost of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the b eta-subunit as an essential residue.