Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Journal of the Brazilian Chemical Society (Online) |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532019001002105 |
Resumo: | Phosphoenolpyruvate carboxykinase (PEPCK) is a ubiquitous enzyme found in all known groups of organisms, acting in the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of divalent metal ion, and dependent of adenosine 5’-triphosphate (ATP) or guanosine-5’-triphosphate (GTP). PEPCK is an important enzyme in the metabolism of some organisms, such as Trypanosoma cruzi, being suggested as a potential drug target to treat Chagas’ disease. Its catalytic activity is, classically, measured by coupled assays. Herein, a direct assay by liquid chromatography-tandem mass spectrometry (LC-MS/MS) capable of quantifying PEP, in co-elution with OAA by the differentiation obtained by the mass spectra, is reported. The developed assay was used throughout the purification protocol in order to measure the activity of PEPCK of T. cruzi, which was expressed in Escherichia coli. The purified enzyme was kinetically characterized by the developed method with Michaelis-Menten constant (KMapp) values of 96 ± 4 and 275 ± 18 µmol L-1 to OAA and ATP as substrates, respectively. The developed assay was also used for ligand screening and proved to be able to identify very low inhibitions for small molecules (50 µmol L-1). |
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Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase ActivityPEPCK activity assayPEPCK purificationLC-MS/MSPhosphoenolpyruvate carboxykinase (PEPCK) is a ubiquitous enzyme found in all known groups of organisms, acting in the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of divalent metal ion, and dependent of adenosine 5’-triphosphate (ATP) or guanosine-5’-triphosphate (GTP). PEPCK is an important enzyme in the metabolism of some organisms, such as Trypanosoma cruzi, being suggested as a potential drug target to treat Chagas’ disease. Its catalytic activity is, classically, measured by coupled assays. Herein, a direct assay by liquid chromatography-tandem mass spectrometry (LC-MS/MS) capable of quantifying PEP, in co-elution with OAA by the differentiation obtained by the mass spectra, is reported. The developed assay was used throughout the purification protocol in order to measure the activity of PEPCK of T. cruzi, which was expressed in Escherichia coli. The purified enzyme was kinetically characterized by the developed method with Michaelis-Menten constant (KMapp) values of 96 ± 4 and 275 ± 18 µmol L-1 to OAA and ATP as substrates, respectively. The developed assay was also used for ligand screening and proved to be able to identify very low inhibitions for small molecules (50 µmol L-1).Sociedade Brasileira de Química2019-10-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532019001002105Journal of the Brazilian Chemical Society v.30 n.10 2019reponame:Journal of the Brazilian Chemical Society (Online)instname:Sociedade Brasileira de Química (SBQ)instacron:SBQ10.21577/0103-5053.20190079info:eu-repo/semantics/openAccessAmaral,Bruno S. doCorrea,Katia Celina S.Corrêa,Arlene G.Souza,Dulce H. F. deCass,Quezia B.eng2019-10-17T00:00:00Zoai:scielo:S0103-50532019001002105Revistahttp://jbcs.sbq.org.brONGhttps://old.scielo.br/oai/scielo-oai.php||office@jbcs.sbq.org.br1678-47900103-5053opendoar:2019-10-17T00:00Journal of the Brazilian Chemical Society (Online) - Sociedade Brasileira de Química (SBQ)false |
dc.title.none.fl_str_mv |
Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity |
title |
Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity |
spellingShingle |
Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity Amaral,Bruno S. do PEPCK activity assay PEPCK purification LC-MS/MS |
title_short |
Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity |
title_full |
Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity |
title_fullStr |
Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity |
title_full_unstemmed |
Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity |
title_sort |
Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity |
author |
Amaral,Bruno S. do |
author_facet |
Amaral,Bruno S. do Correa,Katia Celina S. Corrêa,Arlene G. Souza,Dulce H. F. de Cass,Quezia B. |
author_role |
author |
author2 |
Correa,Katia Celina S. Corrêa,Arlene G. Souza,Dulce H. F. de Cass,Quezia B. |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
Amaral,Bruno S. do Correa,Katia Celina S. Corrêa,Arlene G. Souza,Dulce H. F. de Cass,Quezia B. |
dc.subject.por.fl_str_mv |
PEPCK activity assay PEPCK purification LC-MS/MS |
topic |
PEPCK activity assay PEPCK purification LC-MS/MS |
description |
Phosphoenolpyruvate carboxykinase (PEPCK) is a ubiquitous enzyme found in all known groups of organisms, acting in the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of divalent metal ion, and dependent of adenosine 5’-triphosphate (ATP) or guanosine-5’-triphosphate (GTP). PEPCK is an important enzyme in the metabolism of some organisms, such as Trypanosoma cruzi, being suggested as a potential drug target to treat Chagas’ disease. Its catalytic activity is, classically, measured by coupled assays. Herein, a direct assay by liquid chromatography-tandem mass spectrometry (LC-MS/MS) capable of quantifying PEP, in co-elution with OAA by the differentiation obtained by the mass spectra, is reported. The developed assay was used throughout the purification protocol in order to measure the activity of PEPCK of T. cruzi, which was expressed in Escherichia coli. The purified enzyme was kinetically characterized by the developed method with Michaelis-Menten constant (KMapp) values of 96 ± 4 and 275 ± 18 µmol L-1 to OAA and ATP as substrates, respectively. The developed assay was also used for ligand screening and proved to be able to identify very low inhibitions for small molecules (50 µmol L-1). |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-10-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532019001002105 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532019001002105 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.21577/0103-5053.20190079 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Sociedade Brasileira de Química |
publisher.none.fl_str_mv |
Sociedade Brasileira de Química |
dc.source.none.fl_str_mv |
Journal of the Brazilian Chemical Society v.30 n.10 2019 reponame:Journal of the Brazilian Chemical Society (Online) instname:Sociedade Brasileira de Química (SBQ) instacron:SBQ |
instname_str |
Sociedade Brasileira de Química (SBQ) |
instacron_str |
SBQ |
institution |
SBQ |
reponame_str |
Journal of the Brazilian Chemical Society (Online) |
collection |
Journal of the Brazilian Chemical Society (Online) |
repository.name.fl_str_mv |
Journal of the Brazilian Chemical Society (Online) - Sociedade Brasileira de Química (SBQ) |
repository.mail.fl_str_mv |
||office@jbcs.sbq.org.br |
_version_ |
1750318182168526848 |