The mode of action of recombinant Mycobacterium tuberculosis shikimate kinase: kinetics and thermodynamics analyses
| Autor: | Igor B. Vasconcelos, Mario Sergio Palma, Leonardo Astolfi Rosado, Luiz Augusto Basso, Rafael Najmanovich, Vincent Frappier, Diógenes Santiago Santos |
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| Přispěvatelé: | Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Universidade Estadual Paulista (Unesp), Université de Sherbrooke |
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Entropy
lcsh:Medicine Gibbs Free Energy Shikimic Acid enzyme purification dissociation constant Biochemistry Physical Chemistry oligomer covalent bond Adenosine Triphosphate Enthalpy Proton transport biophysics enzyme kinetics Drug Discovery binding affinity Biomacromolecule-Ligand Interactions recombinant enzyme lcsh:Science Liquid Chromatography Chromatography Multidisciplinary Chemistry Titrimetry fluorescence spectroscopy physical parameters Enzyme structure Recombinant Proteins Enzymes unclassified drug enzyme activity enzyme structure isothermal titration calorimetry Dissociation constant Phosphotransferases (Alcohol Group Acceptor) Infectious Diseases Thermodynamics Medicine solvation vibration Hydrophobic and Hydrophilic Interactions amino acid Research Article Protein Binding chemical reaction Drugs and Devices Drug Research and Development Stereochemistry ligand binding adenosine triphosphate Calorimetry Shikimate kinase Protein Chemistry thermodynamics Bacterial Proteins complex formation biochemistry controlled study steady state Enzyme kinetics Biology constant pressure heat capacity enzyme substrate complex hydrophobicity Enzyme substrate complex Enzyme Kinetics nonhuman catalysis lcsh:R Proteins Isothermal titration calorimetry Hydrogen Bonding Mycobacterium tuberculosis sequence homology Kinetics Small Molecules molecular interaction amino terminal sequence lcsh:Q Steady state (chemistry) proton transport recombinant shikimate kinase |
| Zdroj: | PLoS ONE, Vol 8, Iss 5, p e61918 (2013) Scopus Repositório Institucional da UNESP Universidade Estadual Paulista (UNESP) instacron:UNESP PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:28Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:48:27Z : No. of bitstreams: 1 2-s2.0-84877093392.pdf: 534027 bytes, checksum: d3d5a098e6362ec541790ad16c13e58c (MD5) Made available in DSpace on 2014-05-27T11:29:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-06 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. 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, RS Programa de Pós-Graduação em Medicina e Ciências da Saúde PUCRS, Porto Alegre, RS Programa de Pós-Graduação em Biologia Celular e Molecular PUCRS, Porto Alegre, RS Laboratório 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 Department of Biochemistry Faculty of Medicine Université de Sherbrooke, Sherbrooke, QC Laboratório 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 |
| Databáze: | OpenAIRE |
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