UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation
The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass...
| Autores: | , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2011 |
| País: | Brasil |
| Institución: | Universidade Estadual Paulista (UNESP) |
| Repositorio: | Repositório Institucional da UNESP |
| Idioma: | inglés |
| OAI Identifier: | oai:repositorio.unesp.br:11449/72298 |
| Acceso en línea: | http://dx.doi.org/10.1016/j.abb.2010.10.019 http://hdl.handle.net/11449/72298 |
| Access Level: | acceso abierto |
| Palabra clave: | Allosteric regulation Antitubercular drug target Cooperative kinetics Pyrimidine metabolism Thermodynamic binding parameters UMPK adenosine triphosphate guanosine triphosphate phosphotransferase pyrimidine unclassified drug uridine 5' monophosphate kinase uridine diphosphate uridine triphosphate amino acid metabolism enzyme kinetics enzyme phosphorylation Escherichia coli gene amplification gene sequence molecular cloning molecular weight Mycobacterium tuberculosis nonhuman priority journal protein expression protein purification Adenosine Triphosphate Allosteric Regulation Amino Acid Sequence Cloning, Molecular Escherichia coli Proteins Genes, Suppressor Guanosine Triphosphate Kinetics Ligands Molecular Sequence Data Molecular Weight Polymerase Chain Reaction Pyrimidines Sequence Alignment Sequence Analysis, DNA Spectrometry, Mass, Electrospray Ionization Transferases Uridine Triphosphate Eukaryota |
| Sumario: | The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved. |
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