Multi-site substrate binding and interplay in barley α-amylase 1

Certain starch hydrolases possess secondary carbohydrate binding sites outside of the active site, suggesting that multisite substrate interactions are functionally signi. cant. In barley a- amylase both Tyr 380, situated on a remote non- catalytic domain, and Tyr 105 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 Tyr 380 and Tyr 105 is important, albeit not essential for binding, degradation, and multiple attack on polysaccharides, while Tyr 105 predominates in oligosaccharide hydrolysis. Additional delicate structure/ function relationships of the secondary site are uncovered using Y380A/ H395A, Y380A, and H395A AMY1 mutants. (c) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.