The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Página do item simplificado
dc.contributor.authorRosado, Leonardo Astolfi
dc.contributor.authorVasconcelos, Igor Bordin
dc.contributor.authorPalma, Mario Sergio [UNESP]
dc.contributor.authorFrappier, Vincent
dc.contributor.authorNajmanovich, Rafael Josef
dc.contributor.authorSantos, Diógenes Santiago
dc.contributor.authorBasso, Luiz Augusto
dc.contributor.institutionPontifícia Universidade Católica do Rio Grande do Sul (PUCRS)
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.contributor.institutionUniversité de Sherbrooke
dc.date.accessioned2014-05-27T11:29:28Z
dc.date.available2014-05-27T11:29:28Z
dc.date.issued2013-05-06
dc.description.abstractTuberculosis 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.en
dc.description.affiliationCentro 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, RS
dc.description.affiliationPrograma de Pós-Graduação em Medicina e Ciências da Saúde PUCRS, Porto Alegre, RS
dc.description.affiliationPrograma de Pós-Graduação em Biologia Celular e Molecular PUCRS, Porto Alegre, RS
dc.description.affiliationLaboratório 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, SP
dc.description.affiliationDepartment of Biochemistry Faculty of Medicine Université de Sherbrooke, Sherbrooke, QC
dc.description.affiliationUnespLaboratório 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, SP
dc.identifierhttp://dx.doi.org/10.1371/journal.pone.0061918
dc.identifier.citationPLoS ONE, v. 8, n. 5, 2013.
dc.identifier.doi10.1371/journal.pone.0061918
dc.identifier.file2-s2.0-84877093392.pdf
dc.identifier.issn1932-6203
dc.identifier.lattes2901888624506535
dc.identifier.scopus2-s2.0-84877093392
dc.identifier.urihttp://hdl.handle.net/11449/75353
dc.identifier.wosWOS:000321390200012
dc.language.isoeng
dc.relation.ispartofPLOS ONE
dc.relation.ispartofjcr2.766
dc.relation.ispartofsjr1,164
dc.rights.accessRightsAcesso aberto
dc.sourceScopus
dc.subjectadenosine triphosphate
dc.subjectamino acid
dc.subjectoligomer
dc.subjectrecombinant enzyme
dc.subjectrecombinant shikimate kinase
dc.subjectunclassified drug
dc.subjectamino terminal sequence
dc.subjectbinding affinity
dc.subjectbiochemistry
dc.subjectbiophysics
dc.subjectcatalysis
dc.subjectchemical reaction
dc.subjectcomplex formation
dc.subjectconstant pressure heat capacity
dc.subjectcontrolled study
dc.subjectcovalent bond
dc.subjectdissociation constant
dc.subjectenzyme activity
dc.subjectenzyme kinetics
dc.subjectenzyme purification
dc.subjectenzyme structure
dc.subjectenzyme substrate complex
dc.subjectfluorescence spectroscopy
dc.subjecthydrophobicity
dc.subjectisothermal titration calorimetry
dc.subjectligand binding
dc.subjectmolecular interaction
dc.subjectMycobacterium tuberculosis
dc.subjectnonhuman
dc.subjectphysical parameters
dc.subjectproton transport
dc.subjectsequence homology
dc.subjectsolvation
dc.subjectsteady state
dc.subjectthermodynamics
dc.subjectvibration
dc.titleThe Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analysesen
dc.typeArtigo
dcterms.licensehttp://www.plos.org/open-access/
unesp.author.lattes2901888624506535
unesp.author.orcid0000-0002-7363-8211[3]
unesp.author.orcid0000-0003-0903-2407[7]
unesp.author.orcid0000-0002-4349-0244[1]
unesp.campusUniversidade Estadual Paulista (Unesp), Instituto de Biociências, Rio Claropt

Arquivos

Pacote Original
Agora exibindo 1 - 1 de 1
Imagem de Miniatura
Nome:
2-s2.0-84877093392.pdf
Tamanho:
521.51 KB
Formato:
Adobe Portable Document Format
Baixar

Coleções

Artigos - Biologia - IBRC