A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+
- Autores
- Faraj, Santiago Enrique; Centeno, Mercedes; Rossi, Rolando Carlos; Montes, Monica Raquel
- Año de publicación
- 2019
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state.
Fil: Faraj, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Centeno, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina - Materia
-
CONFORMATIONAL CHANGE
ENZYME KINETICS
ENZYME MECHANISM
LIGAND BINDING KINETICS
MEMBRANE TRANSPORT
NA,K-ATPASE
PHOSPHORYLATED STATES
RB+ OCCLUSION AND DEOCCLUSION - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/93101
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CONICET Digital (CONICET) |
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A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+Faraj, Santiago EnriqueCenteno, MercedesRossi, Rolando CarlosMontes, Monica RaquelCONFORMATIONAL CHANGEENZYME KINETICSENZYME MECHANISMLIGAND BINDING KINETICSMEMBRANE TRANSPORTNA,K-ATPASEPHOSPHORYLATED STATESRB+ OCCLUSION AND DEOCCLUSIONhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state.Fil: Faraj, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Centeno, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaElsevier Science2019-02-15info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/93101Faraj, Santiago Enrique; Centeno, Mercedes; Rossi, Rolando Carlos; Montes, Monica Raquel; A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1861; 2; 15-2-2019; 355-3650005-2736CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0005273618303195info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2018.10.020info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:40:11Zoai:ri.conicet.gov.ar:11336/93101instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:40:11.965CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+ |
title |
A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+ |
spellingShingle |
A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+ Faraj, Santiago Enrique CONFORMATIONAL CHANGE ENZYME KINETICS ENZYME MECHANISM LIGAND BINDING KINETICS MEMBRANE TRANSPORT NA,K-ATPASE PHOSPHORYLATED STATES RB+ OCCLUSION AND DEOCCLUSION |
title_short |
A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+ |
title_full |
A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+ |
title_fullStr |
A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+ |
title_full_unstemmed |
A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+ |
title_sort |
A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+ |
dc.creator.none.fl_str_mv |
Faraj, Santiago Enrique Centeno, Mercedes Rossi, Rolando Carlos Montes, Monica Raquel |
author |
Faraj, Santiago Enrique |
author_facet |
Faraj, Santiago Enrique Centeno, Mercedes Rossi, Rolando Carlos Montes, Monica Raquel |
author_role |
author |
author2 |
Centeno, Mercedes Rossi, Rolando Carlos Montes, Monica Raquel |
author2_role |
author author author |
dc.subject.none.fl_str_mv |
CONFORMATIONAL CHANGE ENZYME KINETICS ENZYME MECHANISM LIGAND BINDING KINETICS MEMBRANE TRANSPORT NA,K-ATPASE PHOSPHORYLATED STATES RB+ OCCLUSION AND DEOCCLUSION |
topic |
CONFORMATIONAL CHANGE ENZYME KINETICS ENZYME MECHANISM LIGAND BINDING KINETICS MEMBRANE TRANSPORT NA,K-ATPASE PHOSPHORYLATED STATES RB+ OCCLUSION AND DEOCCLUSION |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state. Fil: Faraj, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Centeno, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina |
description |
Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-02-15 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://purl.org/coar/resource_type/c_6501 info:ar-repo/semantics/articulo |
format |
article |
status_str |
publishedVersion |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/11336/93101 Faraj, Santiago Enrique; Centeno, Mercedes; Rossi, Rolando Carlos; Montes, Monica Raquel; A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1861; 2; 15-2-2019; 355-365 0005-2736 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/93101 |
identifier_str_mv |
Faraj, Santiago Enrique; Centeno, Mercedes; Rossi, Rolando Carlos; Montes, Monica Raquel; A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1861; 2; 15-2-2019; 355-365 0005-2736 CONICET Digital CONICET |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0005273618303195 info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2018.10.020 |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Elsevier Science |
publisher.none.fl_str_mv |
Elsevier Science |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
reponame_str |
CONICET Digital (CONICET) |
collection |
CONICET Digital (CONICET) |
instname_str |
Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.name.fl_str_mv |
CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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1844613271598923776 |
score |
13.070432 |