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dc.contributor.authorCapila, Ishan
dc.contributor.authorWu, Yi
dc.contributor.authorRethwisch, David W.
dc.contributor.authorMatte, Allan
dc.contributor.authorCygler, Miroslaw
dc.contributor.authorLinhardt, Robert J.
dc.identifier.citationRole of Arginine 292 in the Catalytic Activity of Chondroitin AC Lyase from Flavobacterium heparinum, I. Capila, Y. Wu, D.W. Rethwisch, A. Matte, M. Cygler, R.J. Linhardt, Biochimica et Biophysica Acta, 1597, 260-270, 2002.
dc.descriptionBiochimica et Biophysica Acta, 1597, 260-270
dc.descriptionNote : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
dc.description.abstractChondroitin AC lyase (chondroitinase EC, an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through beta-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords approximately 10-fold higher K(m) values but no significant change in V(max), consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage.
dc.description.sponsorshipNational Institutes of Health
dc.relation.ispartofThe Linhardt Research Labs Online Collection
dc.relation.ispartofRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
dc.subjectChemistry and chemical biology
dc.subjectChemical and biological engineering
dc.subjectBiomedical engineering
dc.titleRole of Arginine 292 in the Catalytic Activity of Chondroitin AC Lyase from Flavobacterium heparinum
dc.rights.holderIn Copyright : this Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
dc.relation.departmentThe Linhardt Research Labs.
dc.relation.departmentThe Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)

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