Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes
Autor(a) principal: | |
---|---|
Data de Publicação: | 2019 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/11937 |
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) and CO2, in the presence of divalent metal ion, and dependent of ATP or GTP. Their catalytic activity is usually measured by coupled assays with malate or pyruvate kinase/lactate dehydrogenases as preferred second enzyme. Thus, the PEPCK reaction product is converted by the action of a NADH-dependent enzyme, wherein the catalytic reaction is followed by the decrease in the absorbance at 340 nm due to the consumption of NADH. The main drawback in coupled assays is that any interference in the activity of the second enzyme will affect the result of the first enzyme. In addition, in the case of PEPCK, NADH has been reported as inhibitor of some of these enzymes. To overcome these problems, herein we report a direct assay to quantity PEP by liquid chromatography-tandem mass spectrometry (LC-MS). The difficulty related to PEPCK’s substrates and products are their higher hydrophilicity and poor retention in reverse-phase elution mode, which leads to coelution or low peak resolution. In spite of this, PEP was quantified in coelution with OAA by the differentiation obtained by the mass spectra data. The developed method was validated, and all the significant figures were in accordance with the adopted criteria. For that, PEPCK of T. cruzi was expressed as an active enzyme in Escherichia coli and purified by anionic exchanged and affinity chromatography. The method was used, then, to monitor the purification of T. cruzi PEPCK as well as to determine all the kinetics parameters of the purified enzyme. The direct method herein disclosed can be used for others PEPCKs. Moreover, a ligand fishing assay based on the immobilization of PEPCKs to magnetic particles was modulated based on a series of synthetic compounds that were screened towards PEPCKs using the solution activity assay here developed. In searching for selective binders, the affinity-based assay was used to screen four ethanolic extracts of Brazilian Cerrado plants: Qualea grandiflora, Diospyros burchellii, Anadenanthera falcata and Byrsonima coccolobifolia. The chemical characterization of the identified ligands was carried out by means of LC-HRMS analysis. These results are fully discussed. The efforts to produce a PEPCK immobilized capillary enzyme reactor to be used as a bioaffinity column in a flow assay is also presented. |
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Amaral, Bruno Sérgio doCass, Quezia Bezerrahttp://lattes.cnpq.br/9197210255594409Souza, Dulce Helena Ferreira dehttp://lattes.cnpq.br/3428955299526003http://lattes.cnpq.br/2729577875052578bac17bd4-d205-4a3d-bac9-b4260db022bf2019-10-16T14:04:24Z2019-10-16T14:04:24Z2019-08-30AMARAL, Bruno Sérgio do. Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes. 2019. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/11937.https://repositorio.ufscar.br/handle/ufscar/11937Phosphoenolpyruvate carboxykinase (PEPCK) is a ubiquitous enzyme found in all known groups of organisms, acting in the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2, in the presence of divalent metal ion, and dependent of ATP or GTP. Their catalytic activity is usually measured by coupled assays with malate or pyruvate kinase/lactate dehydrogenases as preferred second enzyme. Thus, the PEPCK reaction product is converted by the action of a NADH-dependent enzyme, wherein the catalytic reaction is followed by the decrease in the absorbance at 340 nm due to the consumption of NADH. The main drawback in coupled assays is that any interference in the activity of the second enzyme will affect the result of the first enzyme. In addition, in the case of PEPCK, NADH has been reported as inhibitor of some of these enzymes. To overcome these problems, herein we report a direct assay to quantity PEP by liquid chromatography-tandem mass spectrometry (LC-MS). The difficulty related to PEPCK’s substrates and products are their higher hydrophilicity and poor retention in reverse-phase elution mode, which leads to coelution or low peak resolution. In spite of this, PEP was quantified in coelution with OAA by the differentiation obtained by the mass spectra data. The developed method was validated, and all the significant figures were in accordance with the adopted criteria. For that, PEPCK of T. cruzi was expressed as an active enzyme in Escherichia coli and purified by anionic exchanged and affinity chromatography. The method was used, then, to monitor the purification of T. cruzi PEPCK as well as to determine all the kinetics parameters of the purified enzyme. The direct method herein disclosed can be used for others PEPCKs. Moreover, a ligand fishing assay based on the immobilization of PEPCKs to magnetic particles was modulated based on a series of synthetic compounds that were screened towards PEPCKs using the solution activity assay here developed. In searching for selective binders, the affinity-based assay was used to screen four ethanolic extracts of Brazilian Cerrado plants: Qualea grandiflora, Diospyros burchellii, Anadenanthera falcata and Byrsonima coccolobifolia. The chemical characterization of the identified ligands was carried out by means of LC-HRMS analysis. These results are fully discussed. The efforts to produce a PEPCK immobilized capillary enzyme reactor to be used as a bioaffinity column in a flow assay is also presented.A enzima fosfoenolpiruvato carboxiquinase (PEPCK) é amplamente distribuída nos organismos e atua na conversão reversível do fosfoenolpiruvato (PEP) para oxaloacetato (OAA), na presença de CO2 e um cátion divalente, dependente de ATP ou GTP. A sua atividade catalítica é normalmente monitorada por ensaios acoplados com malato desidrogenase ou piruvato quinase/lactato desidrogenase. Assim, o produto da reação da PEPCK é convertido em outro pela ação de uma enzima NADH-dependente, com o monitoramento do NADH consumido através da diminuição da absorbância a 340 nm. A principal desvantagem dos ensaios acoplados é que qualquer interferência na atividade da segunda enzima afeta a atividade da primeira enzima. Também, o NADH foi reportado como inibidor da PEPCK de alguns organismos. Para superar estes problemas, relatamos aqui um ensaio direto para quantificar o PEP por cromatografia a líquido hifenada à espectrometria de massas (LC-MS). A dificuldade relacionada aos substratos e produtos da PEPCK é sua elevada hidrofilicidade e baixa retenção no modo reverso de eluição, o que leva a coeluição ou baixa resolução de picos. Apesar disso, o PEP foi quantificado em coeluição com OAA através da diferenciação obtida pelos íons no espectro de massas. O método desenvolvido foi qualificado e todos os valores estiveram de acordo com os critérios adotados. Assim, a PEPCK de T. cruzi foi expressa como uma enzima ativa em E. coli e purificada por cromatografia aniônica e de afinidade. O método LC-MS foi utilizado, então, para monitorar a purificação da PEPCK de T. cruzi, bem como para determinar todos os parâmetros cinéticos da enzima purificada. O método direto aqui reportado pode ser usado para outras PEPCKs. Além disso, um ensaio de captura de ligantes, através da imobilização da PEPCK em partículas magnéticas, foi modulado com base em uma série de compostos sintéticos que foram selecionados para PEPCK usando o ensaio de atividade em solução aqui desenvolvido. Na busca por ligantes seletivos, foram realizados ensaios de ligand fishing, baseados em afinidade, para a triagem em quatro extratos etanólicos de plantas brasileiras do cerrado: Qualea grandiflora, Diospyros burchellii, Anadenanthera falcata e Byrsonima coccolobifolia. A caracterização química dos ligantes identificados foi realizada através de análise por LC-HRMS. Esses resultados são totalmente discutidos. Os esforços para produzir um reator capilar de enzima imobilizada com a PEPCK para ser usado como uma coluna de bioafinidade em um ensaio de fluxo também são apresentados.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES: Código do Financiamento 001porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Química - PPGQUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessImobilização de enzimasCromatografia a líquidoTriagem de ligantesEnzyme immobilizationLiquid chromatographyLigand screeningCIENCIAS EXATAS E DA TERRA::QUIMICAFosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantesImmobilized phosphoenolpyruvate carboxykinase: new models of ligands screeninginfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis600600867fc213-f339-49e1-a669-a1e54cf68bf1reponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALTese - Bruno Sérgio do Amaral.pdfTese - Bruno Sérgio do Amaral.pdfVersão final da Teseapplication/pdf5301251https://repositorio.ufscar.br/bitstream/ufscar/11937/1/Tese%20-%20Bruno%20S%c3%a9rgio%20do%20Amaral.pdf9f5034bf464416d3f8c60959ff82d81dMD51Carta de Homologação tese Bruno Amaral.pdfCarta de Homologação tese Bruno Amaral.pdfCarta Comprovante assinada pelo Orientadorapplication/pdf111522https://repositorio.ufscar.br/bitstream/ufscar/11937/2/Carta%20de%20Homologa%c3%a7%c3%a3o%20tese%20Bruno%20Amaral.pdf77c3209b2de9104d08da3a8305661ed1MD52CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufscar.br/bitstream/ufscar/11937/3/license_rdfe39d27027a6cc9cb039ad269a5db8e34MD53TEXTTese - Bruno Sérgio do Amaral.pdf.txtTese - Bruno Sérgio do Amaral.pdf.txtExtracted texttext/plain299666https://repositorio.ufscar.br/bitstream/ufscar/11937/4/Tese%20-%20Bruno%20S%c3%a9rgio%20do%20Amaral.pdf.txt7ee8322f7755abf87db8ef696cdb2812MD54Carta de Homologação tese Bruno Amaral.pdf.txtCarta de Homologação tese Bruno Amaral.pdf.txtExtracted texttext/plain1289https://repositorio.ufscar.br/bitstream/ufscar/11937/6/Carta%20de%20Homologa%c3%a7%c3%a3o%20tese%20Bruno%20Amaral.pdf.txt329b47bb327eae848c5a7a2e9e3ec4faMD56THUMBNAILTese - Bruno Sérgio do Amaral.pdf.jpgTese - Bruno Sérgio do Amaral.pdf.jpgIM Thumbnailimage/jpeg9692https://repositorio.ufscar.br/bitstream/ufscar/11937/5/Tese%20-%20Bruno%20S%c3%a9rgio%20do%20Amaral.pdf.jpg1a6881c1bd13ed2f7e7c1e50524f4549MD55Carta de Homologação tese Bruno Amaral.pdf.jpgCarta de Homologação tese Bruno Amaral.pdf.jpgIM Thumbnailimage/jpeg11745https://repositorio.ufscar.br/bitstream/ufscar/11937/7/Carta%20de%20Homologa%c3%a7%c3%a3o%20tese%20Bruno%20Amaral.pdf.jpg723efbb1c298d2ac4659a09ff5db59cfMD57ufscar/119372023-09-18 18:31:45.234oai:repositorio.ufscar.br:ufscar/11937Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:45Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes |
dc.title.alternative.eng.fl_str_mv |
Immobilized phosphoenolpyruvate carboxykinase: new models of ligands screening |
title |
Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes |
spellingShingle |
Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes Amaral, Bruno Sérgio do Imobilização de enzimas Cromatografia a líquido Triagem de ligantes Enzyme immobilization Liquid chromatography Ligand screening CIENCIAS EXATAS E DA TERRA::QUIMICA |
title_short |
Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes |
title_full |
Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes |
title_fullStr |
Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes |
title_full_unstemmed |
Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes |
title_sort |
Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes |
author |
Amaral, Bruno Sérgio do |
author_facet |
Amaral, Bruno Sérgio do |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/2729577875052578 |
dc.contributor.author.fl_str_mv |
Amaral, Bruno Sérgio do |
dc.contributor.advisor1.fl_str_mv |
Cass, Quezia Bezerra |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/9197210255594409 |
dc.contributor.advisor-co1.fl_str_mv |
Souza, Dulce Helena Ferreira de |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/3428955299526003 |
dc.contributor.authorID.fl_str_mv |
bac17bd4-d205-4a3d-bac9-b4260db022bf |
contributor_str_mv |
Cass, Quezia Bezerra Souza, Dulce Helena Ferreira de |
dc.subject.por.fl_str_mv |
Imobilização de enzimas Cromatografia a líquido Triagem de ligantes |
topic |
Imobilização de enzimas Cromatografia a líquido Triagem de ligantes Enzyme immobilization Liquid chromatography Ligand screening CIENCIAS EXATAS E DA TERRA::QUIMICA |
dc.subject.eng.fl_str_mv |
Enzyme immobilization Liquid chromatography Ligand screening |
dc.subject.cnpq.fl_str_mv |
CIENCIAS EXATAS E DA TERRA::QUIMICA |
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) and CO2, in the presence of divalent metal ion, and dependent of ATP or GTP. Their catalytic activity is usually measured by coupled assays with malate or pyruvate kinase/lactate dehydrogenases as preferred second enzyme. Thus, the PEPCK reaction product is converted by the action of a NADH-dependent enzyme, wherein the catalytic reaction is followed by the decrease in the absorbance at 340 nm due to the consumption of NADH. The main drawback in coupled assays is that any interference in the activity of the second enzyme will affect the result of the first enzyme. In addition, in the case of PEPCK, NADH has been reported as inhibitor of some of these enzymes. To overcome these problems, herein we report a direct assay to quantity PEP by liquid chromatography-tandem mass spectrometry (LC-MS). The difficulty related to PEPCK’s substrates and products are their higher hydrophilicity and poor retention in reverse-phase elution mode, which leads to coelution or low peak resolution. In spite of this, PEP was quantified in coelution with OAA by the differentiation obtained by the mass spectra data. The developed method was validated, and all the significant figures were in accordance with the adopted criteria. For that, PEPCK of T. cruzi was expressed as an active enzyme in Escherichia coli and purified by anionic exchanged and affinity chromatography. The method was used, then, to monitor the purification of T. cruzi PEPCK as well as to determine all the kinetics parameters of the purified enzyme. The direct method herein disclosed can be used for others PEPCKs. Moreover, a ligand fishing assay based on the immobilization of PEPCKs to magnetic particles was modulated based on a series of synthetic compounds that were screened towards PEPCKs using the solution activity assay here developed. In searching for selective binders, the affinity-based assay was used to screen four ethanolic extracts of Brazilian Cerrado plants: Qualea grandiflora, Diospyros burchellii, Anadenanthera falcata and Byrsonima coccolobifolia. The chemical characterization of the identified ligands was carried out by means of LC-HRMS analysis. These results are fully discussed. The efforts to produce a PEPCK immobilized capillary enzyme reactor to be used as a bioaffinity column in a flow assay is also presented. |
publishDate |
2019 |
dc.date.accessioned.fl_str_mv |
2019-10-16T14:04:24Z |
dc.date.available.fl_str_mv |
2019-10-16T14:04:24Z |
dc.date.issued.fl_str_mv |
2019-08-30 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
AMARAL, Bruno Sérgio do. Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes. 2019. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/11937. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/11937 |
identifier_str_mv |
AMARAL, Bruno Sérgio do. Fosfoenolpiruvato carboxiquinase imobilizada: novos modelos de triagem de ligantes. 2019. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/11937. |
url |
https://repositorio.ufscar.br/handle/ufscar/11937 |
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600 600 |
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867fc213-f339-49e1-a669-a1e54cf68bf1 |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ info:eu-repo/semantics/openAccess |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ |
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openAccess |
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Universidade Federal de São Carlos Câmpus São Carlos |
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Programa de Pós-Graduação em Química - PPGQ |
dc.publisher.initials.fl_str_mv |
UFSCar |
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Universidade Federal de São Carlos Câmpus São Carlos |
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