Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase
peer reviewed Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double muta...
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ftorbi:oai:orbi.ulg.ac.be:2268/15167 2024-04-21T07:52:38+00:00 Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase D'Amico, Salvino Gerday, Charles Feller, Georges 2002-11-29 https://orbi.uliege.be/handle/2268/15167 https://orbi.uliege.be/bitstream/2268/15167/1/JBC_2002_SS.pdf https://doi.org/10.1074/jbc.M207253200 en eng Amer Soc Biochemistry Molecular Biology Inc urn:issn:0021-9258 urn:issn:1083-351X https://orbi.uliege.be/handle/2268/15167 info:hdl:2268/15167 https://orbi.uliege.be/bitstream/2268/15167/1/JBC_2002_SS.pdf doi:10.1074/jbc.M207253200 scopus-id:2-s2.0-0037195912 info:pmid:12324460 open access http://purl.org/coar/access_right/c_abf2 info:eu-repo/semantics/openAccess Journal of Biological Chemistry, 277 (48), 46110-46115 (2002-11-29) 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 2002 ftorbi https://doi.org/10.1074/jbc.M207253200 2024-03-27T14:52:24Z peer reviewed Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T-m) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated a-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degreesC as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution. Article in Journal/Newspaper Antarc* Antarctic University of Liège: ORBi (Open Repository and Bibliography) Journal of Biological Chemistry 277 48 46110 46115 |
institution |
Open Polar |
collection |
University of Liège: ORBi (Open Repository and Bibliography) |
op_collection_id |
ftorbi |
language |
English |
topic |
Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire |
spellingShingle |
Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire D'Amico, Salvino Gerday, Charles Feller, Georges Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase |
topic_facet |
Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire |
description |
peer reviewed Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T-m) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated a-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degreesC as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution. |
format |
Article in Journal/Newspaper |
author |
D'Amico, Salvino Gerday, Charles Feller, Georges |
author_facet |
D'Amico, Salvino Gerday, Charles Feller, Georges |
author_sort |
D'Amico, Salvino |
title |
Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase |
title_short |
Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase |
title_full |
Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase |
title_fullStr |
Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase |
title_full_unstemmed |
Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase |
title_sort |
dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase |
publisher |
Amer Soc Biochemistry Molecular Biology Inc |
publishDate |
2002 |
url |
https://orbi.uliege.be/handle/2268/15167 https://orbi.uliege.be/bitstream/2268/15167/1/JBC_2002_SS.pdf https://doi.org/10.1074/jbc.M207253200 |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Journal of Biological Chemistry, 277 (48), 46110-46115 (2002-11-29) |
op_relation |
urn:issn:0021-9258 urn:issn:1083-351X https://orbi.uliege.be/handle/2268/15167 info:hdl:2268/15167 https://orbi.uliege.be/bitstream/2268/15167/1/JBC_2002_SS.pdf doi:10.1074/jbc.M207253200 scopus-id:2-s2.0-0037195912 info:pmid:12324460 |
op_rights |
open access http://purl.org/coar/access_right/c_abf2 info:eu-repo/semantics/openAccess |
op_doi |
https://doi.org/10.1074/jbc.M207253200 |
container_title |
Journal of Biological Chemistry |
container_volume |
277 |
container_issue |
48 |
container_start_page |
46110 |
op_container_end_page |
46115 |
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1796935843173105664 |