Mutational analysis of target enzyme recognition of the beta-trefoil fold barley alpha-amylase/subtilisin inhibitor

Birgit Christine Bønsager, Per K. Nielsen, Maher Abou Hachem, Kenji Fukuda, M. Praetorius-Ibba, Birte Svensson

    Research output: Contribution to journalJournal articlepeer-review

    Abstract

    The barley alpha-amylase/subtilisin inhibitor ( BASI) inhibits alpha-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the alpha-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on k(on), whereas the k(off) values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the K-i values of some mutants on the AMY2-binding side increased 2-3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca2+ at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca2+ had weaker affinity for AMY2 and reduced sensitivity to the Ca2+ modulation of the affinity. These results suggest that the Ca2+ and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases.
    Original languageEnglish
    JournalJournal of Biological Chemistry
    Volume280
    Issue number15
    Pages (from-to)14855-14864
    ISSN0021-9258
    DOIs
    Publication statusPublished - 2005

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