Kinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase
peer reviewed A new microcalorimetric method for recording the kinetic parameters k(cat)/K-m and K-i of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to stud...
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2006
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Online Access: | https://orbi.uliege.be/handle/2268/15376 https://doi.org/10.1016/j.jmb.2006.03.004 |
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ftorbi:oai:orbi.ulg.ac.be:2268/15376 2024-10-13T14:01:41+00:00 Kinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase D'Amico, Salvino Sohier, Jean Feller, Georges 2006-05 https://orbi.uliege.be/handle/2268/15376 https://doi.org/10.1016/j.jmb.2006.03.004 en eng Academic Press Ltd Elsevier Science Ltd urn:issn:0022-2836 urn:issn:1089-8638 https://orbi.uliege.be/handle/2268/15376 info:hdl:2268/15376 doi:10.1016/j.jmb.2006.03.004 info:pmid:16580683 restricted access http://purl.org/coar/access_right/c_16ec info:eu-repo/semantics/restrictedAccess Journal of Molecular Biology, 358 (5), 1296-1304 (2006-05) extremophiles psychrophiles microcalorimetry isothermal titration calorimetry Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire journal article http://purl.org/coar/resource_type/c_6501 info:eu-repo/semantics/article peer reviewed 2006 ftorbi https://doi.org/10.1016/j.jmb.2006.03.004 2024-09-27T07:01:45Z peer reviewed A new microcalorimetric method for recording the kinetic parameters k(cat)/K-m and K-i of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic a-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic a-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic a-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site. (c) 2006 Elsevier Ltd. All rights reserved. Article in Journal/Newspaper Antarc* Antarctic University of Liège: ORBi (Open Repository and Bibliography) Antarctic Journal of Molecular Biology 358 5 1296 1304 |
institution |
Open Polar |
collection |
University of Liège: ORBi (Open Repository and Bibliography) |
op_collection_id |
ftorbi |
language |
English |
topic |
extremophiles psychrophiles microcalorimetry isothermal titration calorimetry Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire |
spellingShingle |
extremophiles psychrophiles microcalorimetry isothermal titration calorimetry Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire D'Amico, Salvino Sohier, Jean Feller, Georges Kinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase |
topic_facet |
extremophiles psychrophiles microcalorimetry isothermal titration calorimetry Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire |
description |
peer reviewed A new microcalorimetric method for recording the kinetic parameters k(cat)/K-m and K-i of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic a-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic a-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic a-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site. (c) 2006 Elsevier Ltd. All rights reserved. |
format |
Article in Journal/Newspaper |
author |
D'Amico, Salvino Sohier, Jean Feller, Georges |
author_facet |
D'Amico, Salvino Sohier, Jean Feller, Georges |
author_sort |
D'Amico, Salvino |
title |
Kinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase |
title_short |
Kinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase |
title_full |
Kinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase |
title_fullStr |
Kinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase |
title_full_unstemmed |
Kinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase |
title_sort |
kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase |
publisher |
Academic Press Ltd Elsevier Science Ltd |
publishDate |
2006 |
url |
https://orbi.uliege.be/handle/2268/15376 https://doi.org/10.1016/j.jmb.2006.03.004 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Journal of Molecular Biology, 358 (5), 1296-1304 (2006-05) |
op_relation |
urn:issn:0022-2836 urn:issn:1089-8638 https://orbi.uliege.be/handle/2268/15376 info:hdl:2268/15376 doi:10.1016/j.jmb.2006.03.004 info:pmid:16580683 |
op_rights |
restricted access http://purl.org/coar/access_right/c_16ec info:eu-repo/semantics/restrictedAccess |
op_doi |
https://doi.org/10.1016/j.jmb.2006.03.004 |
container_title |
Journal of Molecular Biology |
container_volume |
358 |
container_issue |
5 |
container_start_page |
1296 |
op_container_end_page |
1304 |
_version_ |
1812812303573188608 |