2qps
"Sugar tongs" mutant Y380A in complex with acarbose
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OverviewOverview
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.
About this StructureAbout this Structure
2QPS is a Single protein structure of sequence from Hordeum vulgare with as ligand. Active as Alpha-amylase, with EC number 3.2.1.1 Known structural/functional Sites: , and . Full crystallographic information is available from OCA.
ReferenceReference
The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity., Bozonnet S, Jensen MT, Nielsen MM, Aghajari N, Jensen MH, Kramhoft B, Willemoes M, Tranier S, Haser R, Svensson B, FEBS J. 2007 Oct;274(19):5055-67. Epub 2007 Sep 4. PMID:17803687
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