Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1

K.S. Bak-Jensen, G. André, T.E. Gottschalk, G. Paes, V. Tran, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    The role in activity of outer regions in the substrate binding cleft in alpha-amylases is illustrated by mutational analysis of Tyr(105) and Thr(212) localized at subsites - 6 and +4 ( substrate cleavage occurs between subsites -1 and +1) in barley alpha-amylase 1 (AMY1). Tyr(105) is conserved in plant alpha-amylases whereas Thr(212) varies in these and related enzymes. Compared with wild-type AMY1, the subsite -6 mutant Y105A has 140, 15, and <1% activity (k(cat)/K-m) on starch, amylose DP17, and 2-chloro-4-nitrophenyl β-D-maltoheptaoside, whereas T212Y at subsite +4 has 32, 370, and 90% activity, respectively. Thus engineering of aromatic stacking interactions at the ends of the 10-subsite long binding cleft affects activity very differently, dependent on the substrate. Y105A dominates in dual subsite -6/+4 [Y105A/T212(Y/W)] AMY1 mutants having almost retained and low activity on starch and oligosaccharides, respectively. Bond cleavage analysis of oligosaccharide degradation by wild-type and mutant AMY1 supports that Tyr105 is critical for binding at subsite -6. Substrate binding is improved by T212(Y/W) introduced at subsite +4 and the [Y105A/ T212(Y/W)] AMY1 double mutants synergistically enhanced productive binding of the substrate aglycone. The enzymatic properties of the series of AMY1 mutants suggest that longer substrates adopt several binding modes. This is in excellent agreement with computed distinct multiple docking solutions observed for maltododecaose at outer binding areas of AMY1 beyond subsites -3 and +3.
    Original languageEnglish
    JournalJournal of Biological Chemistry
    Volume279
    Pages (from-to)10093-10102
    ISSN0021-9258
    Publication statusPublished - 2004

    Fingerprint Dive into the research topics of 'Tyrosine 105 and threonine 212 at outermost substrate binding subsites -6 and +4 control substrate specificity, oligosaccharide cleavage patterns, and multiple binding modes of barley alpha-amylase 1'. Together they form a unique fingerprint.

    Cite this