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 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.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 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.