SUBUNIT STRUCTURES AND ESSENTIAL AMINO-ACID-RESIDUES OF WHITE KIDNEY BEAN (PHASEOLUS-VULGARIS) ALPHA-AMYLASE INHIBITORS

Both white kidney bean ce-amylase inhibitors WKB 858A (MW 42,000) and WKB 858B (MW 20,000) were composed of two subunits as determined by N-terminal amino acid analysis;by amino acid sequence, by SDS-PAGE and by separation on a chromatofocusing column in 8 M urea. N-Terminal amino acids for Inhibitor WKB 858A were alanine and glycine, with a sequence of H2N-Ala-Glu-Asn-Ala-Gly-Thr-Tyr----COOH for deglycosylated 19,000 MW peptide and H2N-Gly-Asn----COOH for deglycosylated 12,000 MW peptide. N-Terminal amino acids for Inhibitor WKB 858B were alanine and serine, with a sequence of H2N-Ala-Thr-Glu-Thr-Ser----COOH for the deglycosylated 9,000 MW peptide and H2N-Ser-Ala-Val-Gly-Leu-Asp-Phe-Val-Leu-Val-Pro-Val-Gln-Pro-Glu-Ser-Lys-Gly-Asp-Thr-Val-Thr-Val-Glu-Phe-Asp----COOH for the deglycosylated 15,000 MW peptide. Chemical modification of 2 of 7 His residues with diethylpyrocarbonate resulted in 26% loss of inhibitory activity. Modification of 1.5 of 7 Trp residues with N-bromosuccinimide gave 60% loss of inhibitory activity. Modification of 2 of 6 Tyr residues with N-acetylimidazole gave 60% loss of inhibitory activity. Modification of 3.6 of 6 Arg residues with p-hydroxyphenylglyoxal gave 64% loss of inhibitor activity. These results indicate the possible importance of one or more His, Trp, Tyr and perhaps Arg residues for inhibitory activity against porcine pancreatic alpha-amylase.