The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1371/journal.pone.0061918 http://hdl.handle.net/11449/75353 |
Resumo: | Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al. |
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The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analysesadenosine triphosphateamino acidoligomerrecombinant enzymerecombinant shikimate kinaseunclassified drugamino terminal sequencebinding affinitybiochemistrybiophysicscatalysischemical reactioncomplex formationconstant pressure heat capacitycontrolled studycovalent bonddissociation constantenzyme activityenzyme kineticsenzyme purificationenzyme structureenzyme substrate complexfluorescence spectroscopyhydrophobicityisothermal titration calorimetryligand bindingmolecular interactionMycobacterium tuberculosisnonhumanphysical parametersproton transportsequence homologysolvationsteady statethermodynamicsvibrationTuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF) Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB) Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RSPrograma de Pós-Graduação em Medicina e Ciências da Saúde PUCRS, Porto Alegre, RSPrograma de Pós-Graduação em Biologia Celular e Molecular PUCRS, Porto Alegre, RSLaboratório de Biologia Estrutural e Zooquímica, Centro de Estudos de Insetos Sociais Departamento de Biologia, Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP), Rio Claro, SPDepartment of Biochemistry Faculty of Medicine Université de Sherbrooke, Sherbrooke, QCLaboratório de Biologia Estrutural e Zooquímica, Centro de Estudos de Insetos Sociais Departamento de Biologia, Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP), Rio Claro, SPPontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Universidade Estadual Paulista (Unesp)Université de SherbrookeRosado, Leonardo AstolfiVasconcelos, Igor BordinPalma, Mario Sergio [UNESP]Frappier, VincentNajmanovich, Rafael JosefSantos, Diógenes SantiagoBasso, Luiz Augusto2014-05-27T11:29:28Z2014-05-27T11:29:28Z2013-05-06info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://dx.doi.org/10.1371/journal.pone.0061918PLoS ONE, v. 8, n. 5, 2013.1932-6203http://hdl.handle.net/11449/7535310.1371/journal.pone.0061918WOS:0003213902000122-s2.0-848770933922-s2.0-84877093392.pdf2901888624506535Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengPLOS ONE2.7661,164info:eu-repo/semantics/openAccess2023-10-07T06:08:23Zoai:repositorio.unesp.br:11449/75353Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T14:17:03.081534Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses |
title |
The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses |
spellingShingle |
The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses Rosado, Leonardo Astolfi adenosine triphosphate amino acid oligomer recombinant enzyme recombinant shikimate kinase unclassified drug amino terminal sequence binding affinity biochemistry biophysics catalysis chemical reaction complex formation constant pressure heat capacity controlled study covalent bond dissociation constant enzyme activity enzyme kinetics enzyme purification enzyme structure enzyme substrate complex fluorescence spectroscopy hydrophobicity isothermal titration calorimetry ligand binding molecular interaction Mycobacterium tuberculosis nonhuman physical parameters proton transport sequence homology solvation steady state thermodynamics vibration |
title_short |
The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses |
title_full |
The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses |
title_fullStr |
The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses |
title_full_unstemmed |
The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses |
title_sort |
The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses |
author |
Rosado, Leonardo Astolfi |
author_facet |
Rosado, Leonardo Astolfi Vasconcelos, Igor Bordin Palma, Mario Sergio [UNESP] Frappier, Vincent Najmanovich, Rafael Josef Santos, Diógenes Santiago Basso, Luiz Augusto |
author_role |
author |
author2 |
Vasconcelos, Igor Bordin Palma, Mario Sergio [UNESP] Frappier, Vincent Najmanovich, Rafael Josef Santos, Diógenes Santiago Basso, Luiz Augusto |
author2_role |
author author author author author author |
dc.contributor.none.fl_str_mv |
Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) Universidade Estadual Paulista (Unesp) Université de Sherbrooke |
dc.contributor.author.fl_str_mv |
Rosado, Leonardo Astolfi Vasconcelos, Igor Bordin Palma, Mario Sergio [UNESP] Frappier, Vincent Najmanovich, Rafael Josef Santos, Diógenes Santiago Basso, Luiz Augusto |
dc.subject.por.fl_str_mv |
adenosine triphosphate amino acid oligomer recombinant enzyme recombinant shikimate kinase unclassified drug amino terminal sequence binding affinity biochemistry biophysics catalysis chemical reaction complex formation constant pressure heat capacity controlled study covalent bond dissociation constant enzyme activity enzyme kinetics enzyme purification enzyme structure enzyme substrate complex fluorescence spectroscopy hydrophobicity isothermal titration calorimetry ligand binding molecular interaction Mycobacterium tuberculosis nonhuman physical parameters proton transport sequence homology solvation steady state thermodynamics vibration |
topic |
adenosine triphosphate amino acid oligomer recombinant enzyme recombinant shikimate kinase unclassified drug amino terminal sequence binding affinity biochemistry biophysics catalysis chemical reaction complex formation constant pressure heat capacity controlled study covalent bond dissociation constant enzyme activity enzyme kinetics enzyme purification enzyme structure enzyme substrate complex fluorescence spectroscopy hydrophobicity isothermal titration calorimetry ligand binding molecular interaction Mycobacterium tuberculosis nonhuman physical parameters proton transport sequence homology solvation steady state thermodynamics vibration |
description |
Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013-05-06 2014-05-27T11:29:28Z 2014-05-27T11:29:28Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1371/journal.pone.0061918 PLoS ONE, v. 8, n. 5, 2013. 1932-6203 http://hdl.handle.net/11449/75353 10.1371/journal.pone.0061918 WOS:000321390200012 2-s2.0-84877093392 2-s2.0-84877093392.pdf 2901888624506535 |
url |
http://dx.doi.org/10.1371/journal.pone.0061918 http://hdl.handle.net/11449/75353 |
identifier_str_mv |
PLoS ONE, v. 8, n. 5, 2013. 1932-6203 10.1371/journal.pone.0061918 WOS:000321390200012 2-s2.0-84877093392 2-s2.0-84877093392.pdf 2901888624506535 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
PLOS ONE 2.766 1,164 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
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1808128340191608832 |