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dc.contributor.authorWipa Sugintaen_US
dc.contributor.authorChomphunuch Songsiriritthigulen_US
dc.contributor.authorArchara Kobdajen_US
dc.contributor.authorRodjana Opassirien_US
dc.contributor.authorJisnuson Svastien_US
dc.contributor.otherSuranaree University of Technologyen_US
dc.contributor.otherNational Synchrotron Research Center, Thailanden_US
dc.contributor.otherMahidol Universityen_US
dc.identifier.citationBiochimica et Biophysica Acta - General Subjects. Vol.1770, No.8 (2007), 1151-1160en_US
dc.description.abstractPoint mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc]2and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired Kmand kcatvalues of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4-G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2-G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1-G5, and then the high-Mrintermediates (G3-G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates. © 2007 Elsevier B.V. All rights reserved.en_US
dc.rightsMahidol Universityen_US
dc.subjectBiochemistry, Genetics and Molecular Biologyen_US
dc.titleMutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase Aen_US
Appears in Collections:Scopus 2006-2010

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