Mapping of barley α-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Abstract

Subsite affinity maps of long substrate binding clefts in barley α-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3–12, revealed unfavorable binding energies at the internal subsites −3 and −5 and at subsites −8 and +3/+4 defining these subsites as binding barriers. Barley α-amylase 1 mutants Y105A and T212Y at subsite −6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (−2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in α-amylases.