This review on barley a-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

a-amylases revealed 6 glycone and 4 aglycone substrate subsites. Moreover, two maltooligosaccharide surface binding

-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 Tyr380 which has no effect on the DMA,

but proved critical for ß-cyclodextrin binding as shown by mutational and surface plasmon resonance analyses. Accordingly,

AMY2 has lower affinity for ß-cyclodextrin. Hydrolysis of amylopectin proceeds via a fast and a slow reaction rate, with

ß-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr380 in activity on amylopectin. Barley seeds produce

-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr380 in activity on amylopectin. Barley seeds produce

different proteinaceous inhibitors acting specifically on insect, animal or plant a-amylases. Rational mutagenesis of barley

a-amylase/subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the

-amylase/subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the

importance of ionic bonds for inhibitory activity.