Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase

Autores
Sosa, Máximo Hernán; Giordana, Lucila; Nowicki, Cristina
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj-PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj-PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj-PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn2+, and HCO3-, being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn2+. Therefore in Lmj-PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj-PEPCK.
Fil: Sosa, Máximo Hernán. 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: Giordana, Lucila. 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: Nowicki, Cristina. 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
3-Mercaptopicolinic Acid
Leishmania Parasites
Mutagenesis Studies
Phosphoenolyruvate Carboxykinase
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/38759
Acceder
id CONICETDig_56232b43b05fb023b52b6cd50076db43
oai_identifier_str oai:ri.conicet.gov.ar:11336/38759
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinaseSosa, Máximo HernánGiordana, LucilaNowicki, Cristina3-Mercaptopicolinic AcidLeishmania ParasitesMutagenesis StudiesPhosphoenolyruvate Carboxykinasehttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj-PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj-PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj-PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn2+, and HCO3-, being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn2+. Therefore in Lmj-PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj-PEPCK.Fil: Sosa, Máximo Hernán. 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: Giordana, Lucila. 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: Nowicki, Cristina. 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 Science Inc2015-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/38759Sosa, Máximo Hernán; Giordana, Lucila; Nowicki, Cristina; Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase; Elsevier Science Inc; Archives of Biochemistry and Biophysics; 583; 10-2015; 120-1290003-9861CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0003986115300187info:eu-repo/semantics/altIdentifier/doi/10.1016/j.abb.2015.07.015info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-11-26T08:55:22Zoai:ri.conicet.gov.ar:11336/38759instacron: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:34982025-11-26 08:55:22.451CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase
title Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase
spellingShingle Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase
Sosa, Máximo Hernán
3-Mercaptopicolinic Acid
Leishmania Parasites
Mutagenesis Studies
Phosphoenolyruvate Carboxykinase
title_short Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase
title_full Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase
title_fullStr Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase
title_full_unstemmed Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase
title_sort Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase
dc.creator.none.fl_str_mv Sosa, Máximo Hernán
Giordana, Lucila
Nowicki, Cristina
author Sosa, Máximo Hernán
author_facet Sosa, Máximo Hernán
Giordana, Lucila
Nowicki, Cristina
author_role author
author2 Giordana, Lucila
Nowicki, Cristina
author2_role author
author
dc.subject.none.fl_str_mv 3-Mercaptopicolinic Acid
Leishmania Parasites
Mutagenesis Studies
Phosphoenolyruvate Carboxykinase
topic 3-Mercaptopicolinic Acid
Leishmania Parasites
Mutagenesis Studies
Phosphoenolyruvate Carboxykinase
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj-PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj-PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj-PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn2+, and HCO3-, being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn2+. Therefore in Lmj-PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj-PEPCK.
Fil: Sosa, Máximo Hernán. 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: Giordana, Lucila. 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: Nowicki, Cristina. 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 This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj-PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj-PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj-PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn2+, and HCO3-, being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn2+. Therefore in Lmj-PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj-PEPCK.
publishDate 2015
dc.date.none.fl_str_mv 2015-10
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/11336/38759
Sosa, Máximo Hernán; Giordana, Lucila; Nowicki, Cristina; Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase; Elsevier Science Inc; Archives of Biochemistry and Biophysics; 583; 10-2015; 120-129
0003-9861
CONICET Digital
CONICET
url http://hdl.handle.net/11336/38759
identifier_str_mv Sosa, Máximo Hernán; Giordana, Lucila; Nowicki, Cristina; Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase; Elsevier Science Inc; Archives of Biochemistry and Biophysics; 583; 10-2015; 120-129
0003-9861
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0003986115300187
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.abb.2015.07.015
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science Inc
publisher.none.fl_str_mv Elsevier Science Inc
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
_version_ 1849873093101617152
score 13.011256

Publicaciones similares