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

• Published in: Archives of biochemistry and biophysics 2011-01, Vol.505 (2), p.202-212
• Main Authors: Rostirolla, Diana C., Breda, Ardala, Rosado, Leonardo A., Palma, Mario S., Basso, Luiz A., Santos, Diógenes S.
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Adenosine Triphosphate - metabolism; Allosteric Regulation; Amino Acid Sequence; amino acids; Antitubercular drug target; calorimetry; catalytic activity; Cloning, Molecular; Cooperative kinetics; DNA; electrospray ionization mass spectrometry; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; gel chromatography; Genes, Suppressor; guanosine triphosphate; Guanosine Triphosphate - metabolism; Kinetics; Ligands; mechanism of action; metabolism; Molecular Sequence Data; Molecular Weight; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - metabolism; phosphorylation; Polymerase Chain Reaction; Pyrimidine metabolism; Pyrimidines - metabolism; RNA precursors; Sequence Alignment; Sequence Analysis, DNA; Spectrometry, Mass, Electrospray Ionization; Thermodynamic binding parameters; titration; Transferases - chemistry; Transferases - genetics; Transferases - isolation & purification; Transferases - metabolism; UMPK; uridine; uridine diphosphate; uridine monophosphate; uridine triphosphate; Uridine Triphosphate - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2010.10.019

► Mycobacterium tuberculosis pyrH-encoded uridine 5′-monophosphate kinase (MtUMPK) is a tetramer. ► MtUMPK catalyzes phosphorylation of UMP to UDP, using ATP–Mg2+ as phosphate donor. ► MtUMPK exhibits positive cooperative kinetics towards ATP. ► GTP and UTP are, respectively, positive and negative effectors of MtUMPK. ► MtUMPK follows a sequential ordered mechanism, in which ATP binds first. 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–Mg2+ 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.