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
- Bak-Jensen, Kristian Sass
- André, Gwenaëlle
- Gottschalk, Tine E.
- Paës, Gabriel
- Tran, Vinh
- Svensson, Birte
DOIAbstract
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 sub...
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