Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis
Ano de defesa: | 2016 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | |
Tipo de documento: | Tese |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Pontif?cia Universidade Cat?lica do Rio Grande do Sul
|
Programa de Pós-Graduação: |
Programa de P?s-Gradua??o em Medicina e Ci?ncias da Sa?de
|
Departamento: |
Escola de Medicina
|
País: |
Brasil
|
Palavras-chave em Português: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://tede2.pucrs.br/tede2/handle/tede/7307 |
Resumo: | The cellular milieu is a complex and crowded aqueous solution. It is thus expected that this large concentration of macromolecules causes deviations from solution ideality. To mimic the intracellular environment, crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (Km, kcat, and kcat/Km) for the chemical reaction catalyzed by 2-trans-enoyl-ACP (CoA) from Mycobacterium tuberculosis (InhA). The results showed that ficoll 70, ficoll 400, and dextran 70 had negligible effects on InhA activity in the range of concentrations used. On the other hand, a complex effect was observed for PEG 6000. Sucrose, which was employed in control experiments, decreased both the kcat/Km values for NADH and kcat for 2-trans-dodecenoyl-CoA (DD-CoA) substrate in a concentration-dependent manner. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution, which likely accounts for the steady-state kinetic results. The presence of crowding agents appears to alter the relative abundance of different conformers of InhA in solution. The effects of the crowding agents on the energy (Ea and E?), enthalpy (?H#), entropy (?S#), and Gibbs free energy (?G#) of activation were determined. The ?G# values for all crowding agents tested were similar to dilute buffer, suggesting that excluded volume effects did not facilitate stable activated ES# complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that "soft" interactions may play a role in macromolecular crowding effects. |
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Basso, Luiz Augusto293.924.890-72http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788585Z8004.799.490-85http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4456070Z2Rotta, Mariane2017-05-26T17:50:17Z2016-12-19http://tede2.pucrs.br/tede2/handle/tede/7307The cellular milieu is a complex and crowded aqueous solution. It is thus expected that this large concentration of macromolecules causes deviations from solution ideality. To mimic the intracellular environment, crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (Km, kcat, and kcat/Km) for the chemical reaction catalyzed by 2-trans-enoyl-ACP (CoA) from Mycobacterium tuberculosis (InhA). The results showed that ficoll 70, ficoll 400, and dextran 70 had negligible effects on InhA activity in the range of concentrations used. On the other hand, a complex effect was observed for PEG 6000. Sucrose, which was employed in control experiments, decreased both the kcat/Km values for NADH and kcat for 2-trans-dodecenoyl-CoA (DD-CoA) substrate in a concentration-dependent manner. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution, which likely accounts for the steady-state kinetic results. The presence of crowding agents appears to alter the relative abundance of different conformers of InhA in solution. The effects of the crowding agents on the energy (Ea and E?), enthalpy (?H#), entropy (?S#), and Gibbs free energy (?G#) of activation were determined. The ?G# values for all crowding agents tested were similar to dilute buffer, suggesting that excluded volume effects did not facilitate stable activated ES# complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that "soft" interactions may play a role in macromolecular crowding effects.O meio intracelular ? uma solu??o aquosa complexa, pois ? preenchida por diversos tipos de macromol?culas. Espera-se que essa grande concentra??o de macromol?culas resulte em um comportamento n?o ideal para a solu??o. Para mimetizar o ambiente intracelular, os efeitos do da ocupa??o macromolecular s?o comumente estudados in vitro utilizando agentes de crowding. O objetivo central do presente estudo ? avaliar os poss?veis efeitos de agentes de crowding macromolecular sint?ticos nos par?metros cin?ticos aparentes de estado-estacion?rio (Km, kcat e kcat/Km) para a rea??o qu?mica catalisada pela 2-trans-enoil-ACP(CoA) redutase de Mycobacterium tuberculosis (InhA). Os resultados mostraram que ficoll 70, ficoll 400 e dextran 70 t?m efeitos negligenci?veis na atividade da InhA na faixa de concentra??o utilizada. Por outro lado, um complexo efeito foi observado na presen?a do PEG 6000. A sacarose, que foi utilizada como controle nos experimentos, reduziu os valores de kcat/Km para o NADH e kcat para o 2-trans-dodecenoil-CoA de uma maneira concentra??o-dependente. Os resultados de din?mica molecular sugerem que a InhA adota uma forma mais compacta na presen?a de sacarose, o que provavelmente tem efeitos nos resultados de cin?tica de estado-estacion?rio. A presen?a dos agentes de crowding parece alterar a abund?ncia relativa dos diferentes conf?rmeros da InhA em solu??o. Os efeitos do crowding macromolecular na energia (Ea e E?), entalpia (?H#), entropia (?S#) e energia livre de Gibbs de ativa??o (?G#) foram determinados. Os valores de ?G# para todos os agentes de crowding testados foram similares ao tamp?o Pipes 100 mM, sugerindo que os efeitos do volume exclu?do n?o facilitam a forma??o do complexo ativado est?vel ES#. A n?o linearidade do gr?fico de Arrhenius para o PEG 6000 sugere que intera??es ?brandas? possam atuar nos efeitos do crowding macromolecular.Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-05-26T17:50:16Z No. of bitstreams: 1 TES_MARIANE_ROTTA_PARCIAL.pdf: 2374056 bytes, checksum: 0b798c90ce9ab78e6edbfeb524d79d97 (MD5)Made available in DSpace on 2017-05-26T17:50:17Z (GMT). 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dc.title.por.fl_str_mv |
Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis |
title |
Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis |
spellingShingle |
Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis Rotta, Mariane 2-trans-enoil-ACP(CoA) Redutase Crowding Macromolecular Efeitos Volume Exclu?do Termodin?mica Din?mica Molecular CIENCIAS DA SAUDE::MEDICINA |
title_short |
Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis |
title_full |
Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis |
title_fullStr |
Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis |
title_full_unstemmed |
Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis |
title_sort |
Efeitos do crowding macromolecular na atividade enzim?tica da 2-trans-ENOIL-ACP (COA) redutaze de Mycobacterium tuberculosis |
author |
Rotta, Mariane |
author_facet |
Rotta, Mariane |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Basso, Luiz Augusto |
dc.contributor.advisor1ID.fl_str_mv |
293.924.890-72 |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788585Z8 |
dc.contributor.authorID.fl_str_mv |
004.799.490-85 |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4456070Z2 |
dc.contributor.author.fl_str_mv |
Rotta, Mariane |
contributor_str_mv |
Basso, Luiz Augusto |
dc.subject.por.fl_str_mv |
2-trans-enoil-ACP(CoA) Redutase Crowding Macromolecular Efeitos Volume Exclu?do Termodin?mica Din?mica Molecular |
topic |
2-trans-enoil-ACP(CoA) Redutase Crowding Macromolecular Efeitos Volume Exclu?do Termodin?mica Din?mica Molecular CIENCIAS DA SAUDE::MEDICINA |
dc.subject.cnpq.fl_str_mv |
CIENCIAS DA SAUDE::MEDICINA |
description |
The cellular milieu is a complex and crowded aqueous solution. It is thus expected that this large concentration of macromolecules causes deviations from solution ideality. To mimic the intracellular environment, crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (Km, kcat, and kcat/Km) for the chemical reaction catalyzed by 2-trans-enoyl-ACP (CoA) from Mycobacterium tuberculosis (InhA). The results showed that ficoll 70, ficoll 400, and dextran 70 had negligible effects on InhA activity in the range of concentrations used. On the other hand, a complex effect was observed for PEG 6000. Sucrose, which was employed in control experiments, decreased both the kcat/Km values for NADH and kcat for 2-trans-dodecenoyl-CoA (DD-CoA) substrate in a concentration-dependent manner. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution, which likely accounts for the steady-state kinetic results. The presence of crowding agents appears to alter the relative abundance of different conformers of InhA in solution. The effects of the crowding agents on the energy (Ea and E?), enthalpy (?H#), entropy (?S#), and Gibbs free energy (?G#) of activation were determined. The ?G# values for all crowding agents tested were similar to dilute buffer, suggesting that excluded volume effects did not facilitate stable activated ES# complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that "soft" interactions may play a role in macromolecular crowding effects. |
publishDate |
2016 |
dc.date.issued.fl_str_mv |
2016-12-19 |
dc.date.accessioned.fl_str_mv |
2017-05-26T17:50:17Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
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publishedVersion |
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http://tede2.pucrs.br/tede2/handle/tede/7307 |
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http://tede2.pucrs.br/tede2/handle/tede/7307 |
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por |
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por |
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7620745074616285884 |
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600 600 600 600 |
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-224747486637135387 |
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info:eu-repo/semantics/openAccess |
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Pontif?cia Universidade Cat?lica do Rio Grande do Sul |
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Programa de P?s-Gradua??o em Medicina e Ci?ncias da Sa?de |
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PUCRS |
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Brasil |
dc.publisher.department.fl_str_mv |
Escola de Medicina |
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Pontif?cia Universidade Cat?lica do Rio Grande do Sul |
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