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

L. Kandra, Maher Abou Hachem, G. Gyemant, B. Kramhoft, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    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 a-amylase I 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 a-amylases.
    Original languageEnglish
    JournalFEBS Letters
    Volume580
    Issue number21
    Pages (from-to)5049-5053
    ISSN0014-5793
    Publication statusPublished - 2006

    Keywords

    • bond cleavage frequencies
    • 2-chloro-4-nitrophenyl
    • beta-D-maltooligosaccharides
    • barley alpha-amylase
    • subsite maps
    • subsite mutants

    Fingerprint Dive into the research topics of 'Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft'. Together they form a unique fingerprint.

    Cite this