Interactions of barley α-amylase isozymes with Ca2 + , substrates and proteinaceous inhibitors

Abstract

α-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/α)8-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca2 + , different substrates, and proteinaceous inhibitors for α-amylases. Isozyme specific effects of Ca2 + on the 80% sequence identical barley α-amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca2 + with identical ligands. A fully hydrated fourth Ca2 + at the interface of the AMY2/barley α-amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca2 + occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca2 + . Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered ‘sugar tongs’ site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley α-amylases are reported in this review.