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Binding of carbohydrates and protein inhibitors to the surface of α-amylases

This paper is available in a repository.
This paper is available in a repository.

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Abstract

This review on barley α-amylases 1 (AMY1) and 2 (AMY2) addresses rational mutations at distal subsites to the catalytic site, polysaccharide hydrolysis, and interactions with proteinaceous inhibitors. Subsite mapping of barley α-amylases revealed 6 glycone and 4 aglycone substrate subsites. Moreover, two maltooligosaccharide surface binding sites have been identified. Engineering of outer subsites -6 and +4 alters action patterns and relative specificities. Thus, compared to wild-type, Y105A AMY1 (subsite -6) shows 140%, 15%, and <1% and T212Y (subsite +4) 32%, 370%, and 90% activity towards starch, maltodextrin, and maltoheptaoside, respectively. The enzyme kinetic properties and modeled maltododecaose complexes suggest binding mode multiplicity. Following an initial hydrolytic cleavage of amylose, an average of 1.9 bonds are cleaved per enzyme-substrate encounter, defining a degree of multiple attack (DMA) of 1.9. DMA increased to 3.3 for Y105A and decreased to 1-1.7 for other subsite mutants. The fusion of a starch-binding domain to AMY1 raised the DMA to 3.0 and increased the amount of higher oligosaccharide products. Remarkably, the subsite mutants had unchanged distribution of released oligosaccharides of DP 5-9, but the profiles differed for the shorter products. A recently identified surface binding site, found exclusively in AMY1, involves the conserved Tyr 380 which has no effect on the DMA, but proved critical for β-cyclodextrin binding as shown by mutational and surface plasmon resonance analyses. Accordingly, AMY2 has lower affinity for β-cyclodextrin. Hydrolysis of amylopectin proceeds via a fast and a slow reaction rate, with β-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr 380 in activity on amylopectin. Barley seeds produce different proteinaceous inhibitors acting specifically on insect, animal or plant α-amylases. Rational mutagenesis of barley α-amylase/ subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the importance of ionic bonds for inhibitory activity.