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 b y separation on a chromatofocusing column in 8 M urea. N-Terminal amin o acids for Inhibitor WKB 858A were alanine and glycine, with a sequen ce of H2N-Ala-Glu-Asn-Ala-Gly-Thr-Tyr----COOH for deglycosylated 19,00 0 MW peptide and H2N-Gly-Asn----COOH for deglycosylated 12,000 MW pept ide. N-Terminal amino acids for Inhibitor WKB 858B were alanine and se rine, with a sequence of H2N-Ala-Thr-Glu-Thr-Ser----COOH for the degly cosylated 9,000 MW peptide and er-Lys-Gly-Asp-Thr-Val-Thr-Val-Glu-Phe- Asp----COOH for the deglycosylated 15,000 MW peptide. Chemical modific ation of 2 of 7 His residues with diethylpyrocarbonate resulted in 26% loss of inhibitory activity. Modification of 1.5 of 7 Trp residues wi th N-bromosuccinimide gave 60% loss of inhibitory activity. Modificati on of 2 of 6 Tyr residues with N-acetylimidazole gave 60% loss of inhi bitory activity. Modification of 3.6 of 6 Arg residues with p-hydroxyp henylglyoxal gave 64% loss of inhibitor activity. These results indica te the possible importance of one or more His, Trp, Tyr and perhaps Ar g residues for inhibitory activity against porcine pancreatic alpha-am ylase.