Isozyme hybrids within the protruding third loop domain of the barley α‐amylase (β/α)8‐barrel implication for BASI sensitivity and substrate affinity

Abstract
Barley α‐amylase isozymes AMY1 and AMY2 contain three structural domains: a catalytic (β/α)8‐barrel (domain A) with a protruding loop (domain B; residues 89–152) that binds Ca2+, and a small C‐terminal domain. Different parts of domain B secure isozyme specific properties as identified for three AMY1–AMY2 hybrids, obtained by homeologous recombination in yeast, with crossing‐over at residues 112, 116, and 144. The AMY1 regions Val90‐Thr112 and Ala145‐Leu161 thus confer high affinities for the substrates p‐nitrophenyl α‐d‐maltoheptaoside and amylose, respectively. Leu117‐Phe144, and to a lesser degree Ala145‐Leu161, are critical for the stability at low pH characteristic of AMY1 and for the sensitivity to barley α‐amylase/subtilisin inhibitor specific to AMY2.

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