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Substrate binding to the inactive mutants of Streptomyces sp. N174 chitosanase: indirect evaluation from the thermal unfolding experiments
- Source :
- FEBS letters. 411(2-3)
- Publication Year :
- 1997
-
Abstract
- Oligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of d -glucosamine oligosaccharides ((GlcN) n , n =3,4,5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu 22 , is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition ( T m ) of E22Q was found to be 44.4°C at pH 7.0. However, the T m increased upon the addition of (GlcN) n by 1.3°C ( n =3), 2.5°C ( n =4), 5.2°C ( n =5), or 7.6°C ( n =6). No appreciable change in T m was observed when (GlcNAc) 6 was added to E22Q. The effect of (GlcN) n on the thermal stability was examined using the other protein, RNase T1, but the oligosaccharide did not affect T m of the protein. Thus, we concluded that the stabilization effect of (GlcN) n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in T m induced by (GlcN) 6 binding were 2.7°C for E22D and 4.2°C for E22A. In E22D or E22A, interaction with (GlcN) 6 seems to be partly disrupted by a conformational distortion in the catalytic cleft.
- Subjects :
- Protein Folding
Glycoside Hydrolases
Stereochemistry
Biophysics
Oligosaccharides
Biochemistry
Streptomyces
Acetylglucosamine
chemistry.chemical_compound
Oligosaccharide binding
Structural Biology
Glucosamine
Aspartic acid
Genetics
Glucosamine oligosaccharide
Chitosanase
Molecular Biology
chemistry.chemical_classification
Alanine
Thermal unfolding
Binding Sites
biology
Temperature
Tryptophan
Substrate (chemistry)
Cell Biology
Oligosaccharide
biology.organism_classification
carbohydrates (lipids)
Crystallography
Spectrometry, Fluorescence
chemistry
Substrate binding
Mutation
Thermodynamics
Protein Binding
Subjects
Details
- ISSN :
- 00145793
- Volume :
- 411
- Issue :
- 2-3
- Database :
- OpenAIRE
- Journal :
- FEBS letters
- Accession number :
- edsair.doi.dedup.....3a42e6ac5d982077d13374cd87cb85a5