title: The conserved tryptophan-arginine-tyrosine motif of a proteinaceous alpha-amylase inhibitor T-76 from Streptomyces nitrosporeus is important for inhibition of animal alpha-amylases but not for an alpha-amylase from Bacillus sp. no. 195.
abstract: Site-directed mutagenesis of Trp-16, Arg-17, and Tyr-18, which were thought to form a putative active site in proteinaceous alpha-amylase inhibitor T-76 from Streptomyces nitrosporeus for inhibition, was performed. The mutation at the site (W16A, R17A, and Y18A) resulted in a marked decrease in inhibitory activity against all animal alpha-amylases tested. Only the alpha-amylase from Bacillus sp. no. 195 (BAA) remained sensitive to all the constructed mutant inhibitors. A competition between T-76 mutants and the wild-type for porcine pancreatic alpha-amylase (PPA) suggest that the loss of inhibitory activity against PPA in mutant inhibitors was due to the decrease in their binding ability for PPA. T-76 formed a complex with BAA as well as PPA at the stoichiometric ratio of 1:1. A competition between BAA and the PPA/T-76 complex suggests that PPA and BAA might bind to the same region or regions close to each other on the T-76 molecule. These results indicate that the conserved Trp-Arg-Tyr motif of T-76 is involved in the interaction between T-76 and PPA while other amino acid residues seem to be important for the T-76/BAA interaction. Since the BAA-type alpha-amylase is the actual target of the inhibitors from microbes in comparison with animal alpha-amylases, BAA might be a better material than PPA to elucidate the "true" function of proteinaceous alpha-amylase inhibitors.
authors.affiliation: Department of Applied Biological Chemistry, College of Agriculture, and Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Sumitani
authors.forename: N
authors.initials: N
authors.lastname: Hattori
authors.forename: Y
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Y
authors.initials: Y
authors.lastname: Okuda
authors.forename: T
authors.initials: T
authors.lastname: Kawaguchi
authors.forename: M
authors.initials: M
authors.lastname: Arai