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PDBsum entry 2qps
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References listed in PDB file
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Key reference
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Title
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The 'Pair of sugar tongs' Site on the non-Catalytic domain c of barley alpha-Amylase participates in substrate binding and activity.
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Authors
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S.Bozonnet,
M.T.Jensen,
M.M.Nielsen,
N.Aghajari,
M.H.Jensen,
B.Kramhøft,
M.Willemoës,
S.Tranier,
R.Haser,
B.Svensson.
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Ref.
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Febs J, 2007,
274,
5055-5067.
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PubMed id
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Abstract
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Some starch-degrading enzymes accommodate carbohydrates at sites situated at a
certain distance from the active site. In the crystal structure of barley
alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This
site on the non-catalytic domain C in the C-terminal part of the molecule
contains a key residue, Tyr380, which has numerous contacts with the
oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to
beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase
affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M
was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes.
The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2
isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at
the 'sugar tongs' in the structure. Crystal structures of both wild-type and
S378P enzymes, but not the Y380A enzyme, showed binding of the
pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site
also contributed importantly to the adsorption to starch granules, as Kd = 0.47
mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which
moreover catalyzed the release of soluble oligosaccharides from starch granules
with only 10% of the wild-type activity. beta-cyclodextrin both inhibited
binding to and suppressed activity on starch granules for wild-type and S378P
enzymes, but did not affect these properties of Y380A, reflecting the functional
role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced
multiple attack, emphasizing that the 'sugar tongs' participates in multivalent
binding of polysaccharide substrates.
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