title: Multi-site substrate binding and interplay in barley alpha-amylase 1.
abstract: Certain starch hydrolases possess secondary carbohydrate binding sites outside of the active site, suggesting that multi-site substrate interactions are functionally significant. In barley alpha-amylase both Tyr380, situated on a remote non-catalytic domain, and Tyr105 in subsite -6 of the active site cleft are principal carbohydrate binding residues. The dual active site/secondary site mutants Y105A/Y380A and Y105A/Y380M show that each of Tyr380 and Tyr105 is important, albeit not essential for binding, degradation, and multiple attack on polysaccharides, while Tyr105 predominates in oligosaccharide hydrolysis. Additional delicate structure/function relationships of the secondary site are uncovered using Y380A/H395A, Y380A, and H395A AMY1 mutants.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Lyngby, Denmark.
authors.forename: Morten Munch
authors.initials: MM
authors.lastname: Nielsen
authors.forename: Eun-Seong
authors.initials: ES
authors.lastname: Seo
authors.forename: Sophie
authors.initials: S
authors.lastname: Bozonnet
authors.forename: Nushin
authors.initials: N
authors.lastname: Aghajari
authors.forename: Xavier
authors.initials: X
authors.lastname: Robert
authors.forename: Richard
authors.initials: R
authors.lastname: Haser
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D001467:Hordeum / Q000201:enzymology*; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D013213:Starch / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics; D047392:beta-Cyclodextrins / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D047392:beta-Cyclodextrins; D014443:Tyrosine; D013213:Starch; D000516:alpha-Amylases; C031215:betadex