UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

• Autor(a) principal: Rostirolla, Diana C.
• Data de Publicação: 2011
• Outros Autores: Breda, Ardala, Rosado, Leonardo A., Palma, Mario Sergio [UNESP], Basso, Luiz A., Santos, Diógenes S.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://dx.doi.org/10.1016/j.abb.2010.10.019; http://hdl.handle.net/11449/72298
• topic: 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

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.