Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2

Jonas Willum Nielsen, Birte Kramhøft, Sophie Bozonnet, Maher Abou Hachem, Susan Louise Svane Stipp, Birte Svensson, Martin Willemoes

    Research output: Contribution to journalJournal articleResearchpeer-review


    Barley α-amylase isozyme 1 (AMY1, EC contains two surface binding sites, SBS1 and SBS2, involved in the degradation of starch granules. The distinct role of SBS1 and SBS2 remains to be fully understood. Mutational analysis of Tyr-380 situated at SBS2 previously revealed that Tyr-380 is required for binding of the amylose helix mimic, β-cyclodextrin. Also, mutant enzymes altered at position 380 displayed reduced binding to starch granules. Similarly, binding of wild type AMY1 to starch granules was suppressed in the presence of β-cyclodextrin. We investigated the role of SBS2 by comparing kinetic properties of the wild type AMY1 and the Y380A mutant enzyme in hydrolysis of amylopectin, amylose and β-limit dextrin, and the inhibition by β-cyclodextrin. Progress curves of the release of reducing ends revealed a bi-exponential hydrolysis of amylopectin and β-limit dextrin, whereas hydrolysis of amylose progressed mono-exponentially. β-Cyclodextrin, however, inhibited only one of the two reaction rates of amylopectin and β-limit dextrin hydrolysis, whereas hydrolysis of amylose was unaffected. The Y380A enzyme showed no detectable inhibition by β-cyclodextrin but displayed similar kinetics to the inhibited wild type AMY1. These results point to SBS2 as an important binding site in amylopectin depolymerization.
    Original languageEnglish
    JournalArchives of Biochemistry and Biophysics
    Issue number1
    Pages (from-to)1-6
    Publication statusPublished - 2012


    • Starch binding
    • Secondary carbohydrate binding sites
    • Bi-exponential progress curves
    • Carbohydrate degradation
    • Complex substrate enzyme kinetics


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