title: Two sulfhydryl groups near the active site of soybean beta-amylase.
abstract: The less reactive SH groups of soybean beta-amylase, SH4, SH5, and SH6, were modified with p-chloromercuribenzoic acid or N-ethylmaleimide, after the reactive SH groups, SH1, SH2, and SH3, were blocked with 5,5'-dithiobis-(2-nitrobenzoic acid) and cyanide. The enzyme activity decreased, accompanied by the modification of SH4. alpha-Cyclodextrin protected SH4 from the modification more effectively than maltose. The SH4-modified enzyme still bound to glucose, maltose, and alpha-cyclodextrin. SH4 was concerned with neither the catalysis nor substrate binding but its large substituent affected the substrate binding site. The sequencing of the 5-(iodoacetoamidoethyl)-aminoaphthalene-1-sulfonate-labeled peptides showed that SH4, SH5, and SH6 are Cys343, Cys82, and Cys208, respectively. Comparison of the primary structure of beta-amylases also showed that the sequence around SH4 (Cys343), as well as SH2 (Cys95), is strongly conserved between higher plant and bacterial beta-amylases. These results agree with the structure model deduced from X-ray crystallography of soybean beta-amylase.
authors.affiliation: Research Institute for Food Science, Kyoto University, Japan.
authors.forename: B
authors.initials: B
authors.lastname: Mikami
authors.forename: K
authors.initials: K
authors.lastname: Nomura
authors.forename: Y
authors.initials: Y
authors.lastname: Morita
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002729:Chloromercuribenzoates; D018360:Crystallography, X-Ray; D003486:Cyanides; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis*; D001614:beta-Amylase / Q000737:chemistry* / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid
chemical_list: D002729:Chloromercuribenzoates; D003486:Cyanides; D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; D020245:p-Chloromercuribenzoic Acid; D004228:Dithionitrobenzoic Acid; D001614:beta-Amylase; D005033:Ethylmaleimide