Kinetic and thermodynamic characterization of dUTP hydrolysis by Plasmodium falciparum dUTPase.
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 Km values, respectively, were observed when compared with the dUTP.Mg2+ complex. Calculation of the activation energy, E(a), and the thermodynamic activation parameters showed that the energetic barrier was approximately 4-fold higher when Mg2+ was depleted. Other divalent ions such as Co2+ or Mn2+ can substitute the physiological cofactor, however the k(cat) was significantly reduced compared to dUTP.Mg2+. Binding and inhibition by dU, dUMP, dUDP, and alpha,beta-imido-dUTP were analysed by ITC and compared with data obtained by spectrophotometric methods and binding equilibrium studies. Product inhibition (Kip dUMP: 99.34 microM) was insignificant yet Ki values for dUDP and alpha,beta-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, Tm, with increasing salt concentrations. Moreover, the thermal unfolding pathway in the presence of salt fits adequately to an irreversible two-state model (N3-->3D).
Quesada-Soriano
Indalecio
I
Area de Química Física, Facultad de Ciencias Experimentales, Universidad de Almería, La Cañada de San Urbano, 04120 Almería, Spain.
Leal
Isabel
I
Casas-Solvas
Juan M
JM
Vargas-Berenguel
Antonio
A
Barón
Carmen
C
Ruiz-Pérez
Luis M
LM
González-Pacanowska
Dolores
D
García-Fuentes
Luis
L
D000818:Animals; D002151:Calorimetry; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism*; D013816:Thermodynamics
D003856:Deoxyuracil Nucleotides; D015800:Protozoan Proteins; C027078:deoxyuridine triphosphate; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D008274:Magnesium