Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only
Abstract The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kine...
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doaj-4c0f711acda144658dcba1c0d30349c82020-12-08T01:10:46ZengNature Publishing GroupScientific Reports2045-23222017-07-017111410.1038/s41598-017-07266-wObserved crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume onlyMariane Rotta0Luis F. S. M. Timmers1Carlos Sequeiros-Borja2Cristiano V. Bizarro3Osmar N. de Souza4Diogenes S. Santos5Luiz A. Basso6Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Laboratório de Bioinformática, Modelagem e Simulação de Biossistemas (LABIO), Faculdade de Informática, PUCRSLaboratório de Bioinformática, Modelagem e Simulação de Biossistemas (LABIO), Faculdade de Informática, PUCRSInstituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Laboratório de Bioinformática, Modelagem e Simulação de Biossistemas (LABIO), Faculdade de Informática, PUCRSInstituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Abstract The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K m , k cat , and k cat /K m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k cat /K m for NADH and k cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E a and E η ), enthalpy (∆H # ), entropy (∆S # ), and Gibbs free energy (∆G # ) of activation were determined. The ∆G # values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES # complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that “soft” interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only.https://doi.org/10.1038/s41598-017-07266-w |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Mariane Rotta Luis F. S. M. Timmers Carlos Sequeiros-Borja Cristiano V. Bizarro Osmar N. de Souza Diogenes S. Santos Luiz A. Basso |
spellingShingle |
Mariane Rotta Luis F. S. M. Timmers Carlos Sequeiros-Borja Cristiano V. Bizarro Osmar N. de Souza Diogenes S. Santos Luiz A. Basso Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only Scientific Reports |
author_facet |
Mariane Rotta Luis F. S. M. Timmers Carlos Sequeiros-Borja Cristiano V. Bizarro Osmar N. de Souza Diogenes S. Santos Luiz A. Basso |
author_sort |
Mariane Rotta |
title |
Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only |
title_short |
Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only |
title_full |
Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only |
title_fullStr |
Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only |
title_full_unstemmed |
Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only |
title_sort |
observed crowding effects on mycobacterium tuberculosis 2-trans-enoyl-acp (coa) reductase enzyme activity are not due to excluded volume only |
publisher |
Nature Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2017-07-01 |
description |
Abstract The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K m , k cat , and k cat /K m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k cat /K m for NADH and k cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E a and E η ), enthalpy (∆H # ), entropy (∆S # ), and Gibbs free energy (∆G # ) of activation were determined. The ∆G # values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES # complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that “soft” interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only. |
url |
https://doi.org/10.1038/s41598-017-07266-w |
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