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Use of (18)O water and ESI-MS detection in subsite characterisation and investigation of the hydrolytic action of an endoglucanase.

Schagerlöf, Herje LU ; Nilsson, Carina LU ; Gorton, Lo LU ; Tjerneld, Folke LU ; Stålbrand, Henrik LU and Cohen, Arieh (2009) In Analytical and Bioanalytical Chemistry 394(7). p.1977-1984
Abstract
We present a novel method for investigating subsite-substrate interactions of glycoside hydrolases and the determination of the oligosaccharide cleavage point based on the analysis of the hydrolysis products produced in the presence of (18)O-labelled water. Conventional techniques for such determination of the hydrolysis pattern call for the chemical modification of the substrate, whereas the method presented makes it possible to use natural substrates, utilising the selectivity and sensitivity of mass spectrometry. This method is very useful for the detection and analysis of enzyme-catalysed hydrolysis, provided that the conditions are chosen where (18)O incorporation without the presence of the enzyme is absent or undetectable. Such... (More)
We present a novel method for investigating subsite-substrate interactions of glycoside hydrolases and the determination of the oligosaccharide cleavage point based on the analysis of the hydrolysis products produced in the presence of (18)O-labelled water. Conventional techniques for such determination of the hydrolysis pattern call for the chemical modification of the substrate, whereas the method presented makes it possible to use natural substrates, utilising the selectivity and sensitivity of mass spectrometry. This method is very useful for the detection and analysis of enzyme-catalysed hydrolysis, provided that the conditions are chosen where (18)O incorporation without the presence of the enzyme is absent or undetectable. Such conditions were found and used in incubations of cellopentaose with the well-characterised endoglucanase Cel5A from Bacillus agaradhaerens. We were able to confirm that the preferred glycoside bond to be hydrolysed is the third one counting from the non-reducing end of the cellopentaose. Thus, cellopentaose prefers to bind from the -3 to the +2 subsites, which is in accordance with published crystallographic data. The main advantage of the method presented is that there is no need for a priori chemical modification/labelling of oligosaccharide substrates, which are processes that can disturb the enzyme-substrate interaction. From (18)O incorporation we could demonstrate that the enzyme also has an oxygen-exchange activity on cellotriose and cellobiose. This is in agreement with the mechanism for transglycosylation and indicates that it is possible for the enzyme to perform such reactions. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Analytical and Bioanalytical Chemistry
volume
394
issue
7
pages
1977 - 1984
publisher
Springer
external identifiers
  • wos:000268000600028
  • pmid:19543714
  • scopus:68349090366
ISSN
1618-2642
DOI
10.1007/s00216-009-2891-9
language
English
LU publication?
yes
id
6dfe10c4-a3b4-449f-9141-0d3274b1d6fa (old id 1434059)
date added to LUP
2009-07-02 15:41:09
date last changed
2017-08-06 03:33:55
@article{6dfe10c4-a3b4-449f-9141-0d3274b1d6fa,
  abstract     = {We present a novel method for investigating subsite-substrate interactions of glycoside hydrolases and the determination of the oligosaccharide cleavage point based on the analysis of the hydrolysis products produced in the presence of (18)O-labelled water. Conventional techniques for such determination of the hydrolysis pattern call for the chemical modification of the substrate, whereas the method presented makes it possible to use natural substrates, utilising the selectivity and sensitivity of mass spectrometry. This method is very useful for the detection and analysis of enzyme-catalysed hydrolysis, provided that the conditions are chosen where (18)O incorporation without the presence of the enzyme is absent or undetectable. Such conditions were found and used in incubations of cellopentaose with the well-characterised endoglucanase Cel5A from Bacillus agaradhaerens. We were able to confirm that the preferred glycoside bond to be hydrolysed is the third one counting from the non-reducing end of the cellopentaose. Thus, cellopentaose prefers to bind from the -3 to the +2 subsites, which is in accordance with published crystallographic data. The main advantage of the method presented is that there is no need for a priori chemical modification/labelling of oligosaccharide substrates, which are processes that can disturb the enzyme-substrate interaction. From (18)O incorporation we could demonstrate that the enzyme also has an oxygen-exchange activity on cellotriose and cellobiose. This is in agreement with the mechanism for transglycosylation and indicates that it is possible for the enzyme to perform such reactions.},
  author       = {Schagerlöf, Herje and Nilsson, Carina and Gorton, Lo and Tjerneld, Folke and Stålbrand, Henrik and Cohen, Arieh},
  issn         = {1618-2642},
  language     = {eng},
  number       = {7},
  pages        = {1977--1984},
  publisher    = {Springer},
  series       = {Analytical and Bioanalytical Chemistry},
  title        = {Use of (18)O water and ESI-MS detection in subsite characterisation and investigation of the hydrolytic action of an endoglucanase.},
  url          = {http://dx.doi.org/10.1007/s00216-009-2891-9},
  volume       = {394},
  year         = {2009},
}