Journal article
Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum
Biochimica et biophysica acta, Protein structure and molecular enzymology, Vol.1597(2), pp.260-270
2002
DOI: 10.1016/S0167-4838(02)00304-7
PMID: 12044904
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 β-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 ∼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.
Details
- Title: Subtitle
- Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum
- Creators
- Ishan Capila - Department of Medicinal and Natural Products Chemistry, University of Iowa, Iowa City, IA 52242, USAYi Wu - Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, IA 52242, USADavid W Rethwisch - Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, IA 52242, USAAllan Matte - Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2Miroslaw Cygler - Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2Robert J Linhardt - Department of Medicinal and Natural Products Chemistry, University of Iowa, Iowa City, IA 52242, USA
- Resource Type
- Journal article
- Publication Details
- Biochimica et biophysica acta, Protein structure and molecular enzymology, Vol.1597(2), pp.260-270
- DOI
- 10.1016/S0167-4838(02)00304-7
- PMID
- 12044904
- NLM abbreviation
- Biochim Biophys Acta
- ISSN
- 0167-4838
- eISSN
- 1879-2588
- Publisher
- Elsevier B.V
- Language
- English
- Date published
- 2002
- Academic Unit
- Chemical and Biochemical Engineering
- Record Identifier
- 9984004193602771
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