Kinetic and thermodynamic characterization of dUTP hydrolysis by Plasmodium falciparum dUTPase

• Published in: Biochimica et biophysica acta 2008-09, Vol.1784 (9), p.1347-1355
• Main Authors: Quesada-Soriano, Indalecio, Leal, Isabel, Casas-Solvas, Juan M., Vargas-Berenguel, Antonio, Barón, Carmen, Ruiz-Pérez, Luis M., González-Pacanowska, Dolores, García-Fuentes, Luis
• Format: Article
• Language: English
• Subjects: Animals; Binding; Calorimetry; Deoxyuracil Nucleotides - metabolism; dUTPase; Hydrolysis; Kinetic; Kinetics; Magnesium - pharmacology; Nuclear Magnetic Resonance, Biomolecular; Osmolar Concentration; Plasmodium falciparum; Plasmodium falciparum - enzymology; Protozoan Proteins - metabolism; Pyrophosphatases - metabolism; Thermal denaturation; Thermodynamics
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2008.05.014

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and plays an important role in nucleotide metabolism and DNA replication controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. Isothermal titration calorimetry has been applied to the determination of the kinetic and thermodynamic parameters of the trimeric Plasmodium falciparum dUTPase, a potential drug target against malaria. The role of divalent ions in binding, and inhibition by different uridine derivatives has been assessed. When dUTP hydrolysis in the presence of EDTA was evaluated, a 105-fold decrease and a 12-fold increase of the k cat and K m values, respectively, were observed when compared with the dUTP·Mg 2+ complex. Calculation of the activation energy, E a, and the thermodynamic activation parameters showed that the energetic barrier was ~ 4-fold higher when Mg 2+ was depleted. Other divalent ions such as Co 2+ or Mn 2+ can substitute the physiological cofactor, however the k cat was significantly reduced compared to dUTP·Mg 2+. Binding and inhibition by dU, dUMP, dUDP, and α,β-imido-dUTP were analysed by ITC and compared with data obtained by spectrophotometric methods and binding equilibrium studies. Product inhibition ( K ip dUMP: 99.34 μM) was insignificant yet K i values for dUDP and α,β-imido-dUTP were in the low micromolar range. The effect of ionic strength on protein stability was also monitored. DSC analysis evidenced a slight increase in the unfolding temperature, T m, with increasing salt concentrations. Moreover, the thermal unfolding pathway in the presence of salt fits adequately to an irreversible two-state model (N 3 → 3D).