Role of Arginine 292 in the Catalytic Activity of Chondroitin AC Lyase from Flavobacterium heparinum
Authors
Capila, Ishan
Wu, Yi
Rethwisch, David W.
Matte, Allan
Cygler, Miroslaw
Linhardt, Robert J.
ORCID
https://orcid.org/0000-0003-2219-5833
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Other Contributors
Issue Date
2002-06-03
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Degree
Terms of Use
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Full Citation
Role 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.
Abstract
Chondroitin AC lyase (chondroitinase EC 4.2.2.5), 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.
Description
Biochimica et Biophysica Acta, 1597, 260-270
Note : 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.
Note : 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.
Department
The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
Publisher
Relationships
The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
https://harc.rpi.edu/
Rensselaer Polytechnic Institute, Troy, NY
Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
https://harc.rpi.edu/
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