TAILIEUCHUNG - Báo cáo khoa học: The ‘pair of sugar tongs’ site on the non-catalytic domain C of barley a-amylase participates in substrate binding and activity

Some starch-degrading enzymes accommodate carbohydrates at sites situ-ated at a certain distance from the active site. In the crystal structure of barleya-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 mole-cule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. | ỊFEBS Journal The pair of sugar tongs site on the non-catalytic domain C of barley a-amylase participates in substrate binding and activity Sophie Bozonnet1 2 Morten T. Jensen2 Morten M. Nielsen1 Nushin Aghajari3 Malene H. Jensen3 Birte Kramh0ft1 2 Martin Willemoes1 2 Samuel Tranier3 Richard Haser3 and Birte Svensson1 2 1 Enzyme and Protein Chemistry BioCentrum-DTU TechnicalUniversity of Denmark Kgs. Lyngby Denmark 2 Carlsberg Laboratory Valby Denmark 3 Laboratoire de BioCristallographie Institut de Biologie et Chimie des Proteines Universite de Lyon France Keywords barley a-amylase crystal structures secondary carbohydrate-binding sites starch granules surface plasmon resonance Correspondence B. Svensson Enzyme and Protein Chemistry BioCentrum-DTU Technical University of Denmark Soltofts Plads Bldg 224 DK-2800 Kgs. Lyngby Denmark Fax 45 45 88 63 07 Tel 45 45 25 27 40 E-mail bis@ Received 1 June 2007 revised 18 July 2007 accepted 1 August 2007 doi Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley a-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 b-cyclodextrin-Sepharose a starch-mimic resin used for a-amylase affinity purification. The Kd for b-cyclodextrin binding to Y380A and Y380M was mM compared to mM for the wild-type S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley a-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 .

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