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 α-amylase 1
2004
Bak-Jensen , Kristian Sass (Carlsberg Research Center, Copenhagen(Danemark). Carlsberg Laboratory, Department of Chemistry) | André , Gwenaëlle (INRA (France). UR 0783 Physicochimie des Macromolécules) | Gottschalk , Tine E. (INRA (France). UR 0783 Physicochimie des Macromolécules) | Paës , Gabriel (INRA (France). UMR 0614 Fractionnement des Agroressources et Emballage) | Tran , Vinh (INRA (France). UR 0783 Physicochimie des Macromolécules) | Svensson , Birte (Carlsberg Research CenterTechnical University of Denmark, CopenhagenLyngby(Danemark). Carlsberg Laboratory, Department of ChemistryBiochemistry and Nutrition Group, BioCentrum-DTU)
The role in activity of outer regions in the substrate binding cleft in α-amylases is illustrated by mutational analysis of Tyr105 and Thr212 localized at subsites –6 and +4 (substrate cleavage occurs between subsites –1 and +1) in barley α-amylase 1 (AMY1). Tyr105 is conserved in plant α-amylases whereas Thr212 varies in these and related enzymes. Compared with wild-type AMY1, the subsite –6 mutant Y105A has 140, 15, and <1% activity (kcat/Km) 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.
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