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

Lili Kandra, Maher Abou Hachem, Gyöngyi Gyémánt, Birte Kramhøft, Birte Svensson

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)5049-5053
Number of pages5
JournalFEBS letters
Volume580
Issue number21
DOIs
Publication statusPublished - Sep 18 2006

Keywords

  • 2-Chloro-4-nitrophenyl β-d-maltooligosaccharides
  • Barley α-amylase
  • Bond cleavage frequencies
  • Subsite maps
  • Subsite mutants

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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