title: Mapping of barley alpha-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 alpha-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 alpha-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 alpha-amylases.
authors.affiliation: Department of Biochemistry, Faculty of Sciences, University of Debrecen, Debrecen H-4010, Hungary.
authors.forename: Lili
authors.initials: L
authors.lastname: Kandra
authors.forename: Maher Abou
authors.initials: MA
authors.lastname: Hachem
authors.forename: Gyöngyi
authors.initials: G
authors.lastname: Gyémánt
authors.forename: Birte
authors.initials: B
authors.lastname: Kramhøft
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000818:Animals; D001665:Binding Sites; D001704:Biopolymers; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry; D011817:Rabbits; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D007527:Isoenzymes; D050505:Mutant Proteins; D009844:Oligosaccharides; C021705:maltooligosaccharides; D000516:alpha-Amylases