Degradation of the starch components amylopectin and amylose by barley α-amylase 1: role of surface binding site 2.
Barley α-amylase isozyme 1 (AMY1, EC 3.2.1.1) 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.
Nano-Science Center, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Jonas Willum
JW
Nielsen
Birte
B
Kramhøft
Sophie
S
Bozonnet
Maher
M
Abou Hachem
S L S
SL
Stipp
Birte
B
Svensson
Martin
M
Willemoës
D000687:Amylopectin / Q000378:metabolism*; D000688:Amylose / Q000378:metabolism*; D001665:Binding Sites; D001467:Hordeum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D020033:Protein Isoforms / Q000378:metabolism; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000378:metabolism
D020033:Protein Isoforms; D047392:beta-Cyclodextrins; D000688:Amylose; D000687:Amylopectin; D000516:alpha-Amylases; C031215:betadex