Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA

Autores
Incicco, Juan Jeremías; Gebhard, Leopoldo German; González Lebrero, Rodolfo M.; Gamarnik, Andrea V.; Sergio B. Kaufman
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides -evaluated as the ratio (kcat/KM)- was GTP=ATP=CTP > UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat) and a 100 fold decrease in the apparent affinity (KM) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and *20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.
Fil: Incicco, Juan Jeremías. DTO.DE QUIMICA BIOLOGICA; INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS;
Fil: Gebhard, Leopoldo German. INST.DE INVEST.BIOQUIMICAS DE BS.AS(I); FUND.INSTITUTO LELOIR;
Fil: González Lebrero, Rodolfo M.. DTO.DE QUIMICA BIOLOGICA; INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS;
Fil: Andrea V. Gamarnik. INST.DE INVEST.BIOQUIMICAS DE BS.AS(I); FUND.INSTITUTO LELOIR;
Fil: Sergio B. Kaufman. INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS; DTO.DE QUIMICA BIOLOGICA;
Materia
RNA helicase
Nonstructural protein 3
Dengue virus
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/565

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network_name_str CONICET Digital (CONICET)
spelling Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNAIncicco, Juan JeremíasGebhard, Leopoldo GermanGonzález Lebrero, Rodolfo M.Gamarnik, Andrea V.Sergio B. KaufmanRNA helicaseNonstructural protein 3Dengue virushttps://purl.org/becyt/ford/1https://purl.org/becyt/ford/1.6Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides -evaluated as the ratio (kcat/KM)- was GTP=ATP=CTP > UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat) and a 100 fold decrease in the apparent affinity (KM) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and *20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.Fil: Incicco, Juan Jeremías. DTO.DE QUIMICA BIOLOGICA; INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS;Fil: Gebhard, Leopoldo German. INST.DE INVEST.BIOQUIMICAS DE BS.AS(I); FUND.INSTITUTO LELOIR;Fil: González Lebrero, Rodolfo M.. DTO.DE QUIMICA BIOLOGICA; INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS;Fil: Andrea V. Gamarnik. INST.DE INVEST.BIOQUIMICAS DE BS.AS(I); FUND.INSTITUTO LELOIR;Fil: Sergio B. Kaufman. INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS; DTO.DE QUIMICA BIOLOGICA;Public Library Science2013-03-19info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/565Incicco, Juan Jeremías; Gebhard, Leopoldo German; González Lebrero, Rodolfo M. ; Andrea V. Gamarnik; Sergio B. Kaufman; Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA; Public Library Science; Plos One; 8; 3; 19-3-2013; 1-12;1932-6203enginfo:eu-repo/semantics/altIdentifier/url/http://dx.plos.org/10.1371/journal.pone.0058508info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:08:09Zoai:ri.conicet.gov.ar:11336/565instacron: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 10:08:09.785CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA
title Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA
spellingShingle Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA
Incicco, Juan Jeremías
RNA helicase
Nonstructural protein 3
Dengue virus
title_short Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA
title_full Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA
title_fullStr Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA
title_full_unstemmed Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA
title_sort Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA
dc.creator.none.fl_str_mv Incicco, Juan Jeremías
Gebhard, Leopoldo German
González Lebrero, Rodolfo M.
Gamarnik, Andrea V.
Sergio B. Kaufman
author Incicco, Juan Jeremías
author_facet Incicco, Juan Jeremías
Gebhard, Leopoldo German
González Lebrero, Rodolfo M.
Gamarnik, Andrea V.
Sergio B. Kaufman
author_role author
author2 Gebhard, Leopoldo German
González Lebrero, Rodolfo M.
Gamarnik, Andrea V.
Sergio B. Kaufman
author2_role author
author
author
author
dc.subject.none.fl_str_mv RNA helicase
Nonstructural protein 3
Dengue virus
topic RNA helicase
Nonstructural protein 3
Dengue virus
purl_subject.fl_str_mv https://purl.org/becyt/ford/1
https://purl.org/becyt/ford/1.6
dc.description.none.fl_txt_mv Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides -evaluated as the ratio (kcat/KM)- was GTP=ATP=CTP > UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat) and a 100 fold decrease in the apparent affinity (KM) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and *20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.
Fil: Incicco, Juan Jeremías. DTO.DE QUIMICA BIOLOGICA; INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS;
Fil: Gebhard, Leopoldo German. INST.DE INVEST.BIOQUIMICAS DE BS.AS(I); FUND.INSTITUTO LELOIR;
Fil: González Lebrero, Rodolfo M.. DTO.DE QUIMICA BIOLOGICA; INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS;
Fil: Andrea V. Gamarnik. INST.DE INVEST.BIOQUIMICAS DE BS.AS(I); FUND.INSTITUTO LELOIR;
Fil: Sergio B. Kaufman. INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS; DTO.DE QUIMICA BIOLOGICA;
description Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides -evaluated as the ratio (kcat/KM)- was GTP=ATP=CTP > UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat) and a 100 fold decrease in the apparent affinity (KM) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and *20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.
publishDate 2013
dc.date.none.fl_str_mv 2013-03-19
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/11336/565
Incicco, Juan Jeremías; Gebhard, Leopoldo German; González Lebrero, Rodolfo M. ; Andrea V. Gamarnik; Sergio B. Kaufman; Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA; Public Library Science; Plos One; 8; 3; 19-3-2013; 1-12;
1932-6203
url http://hdl.handle.net/11336/565
identifier_str_mv Incicco, Juan Jeremías; Gebhard, Leopoldo German; González Lebrero, Rodolfo M. ; Andrea V. Gamarnik; Sergio B. Kaufman; Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by sRNA; Public Library Science; Plos One; 8; 3; 19-3-2013; 1-12;
1932-6203
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://dx.plos.org/10.1371/journal.pone.0058508
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
dc.publisher.none.fl_str_mv Public Library Science
publisher.none.fl_str_mv Public Library 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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