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

    Cite this

    @article{24a6602121264189ae8b5fe75012ec3d,
    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 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.",
    keywords = "bond cleavage frequencies, 2-chloro-4-nitrophenyl, beta-D-maltooligosaccharides, barley alpha-amylase, subsite maps, subsite mutants",
    author = "L. Kandra and {Abou Hachem}, Maher and G. Gyemant and B. Kramhoft and Birte Svensson",
    year = "2006",
    language = "English",
    volume = "580",
    pages = "5049--5053",
    journal = "F E B S Letters",
    issn = "0014-5793",
    publisher = "JohnWiley & Sons Ltd.",
    number = "21",

    }

    Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft. / Kandra, L.; Abou Hachem, Maher; Gyemant, G.; Kramhoft, B.; Svensson, Birte.

    In: FEBS Letters, Vol. 580, No. 21, 2006, p. 5049-5053.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    AU - Kandra, L.

    AU - Abou Hachem, Maher

    AU - Gyemant, G.

    AU - Kramhoft, B.

    AU - Svensson, Birte

    PY - 2006

    Y1 - 2006

    N2 - 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.

    AB - 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.

    KW - bond cleavage frequencies

    KW - 2-chloro-4-nitrophenyl

    KW - beta-D-maltooligosaccharides

    KW - barley alpha-amylase

    KW - subsite maps

    KW - subsite mutants

    M3 - Journal article

    VL - 580

    SP - 5049

    EP - 5053

    JO - F E B S Letters

    JF - F E B S Letters

    SN - 0014-5793

    IS - 21

    ER -