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 bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase 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-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue.
Takahashi
T
T
Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
Hiramoto
S
S
Wato
S
S
Nishimoto
T
T
Wada
Y
Y
Nagai
K
K
Yamaguchi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D007887:Fabaceae / Q000737:chemistry* / Q000235:genetics; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010946:Plants, Medicinal; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014443:Tyrosine / Q000737:chemistry; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D010940:Plant Proteins; D014443:Tyrosine; D001120:Arginine; D000516:alpha-Amylases