Quesada-Soriano Indalecio, Leal Isabel, Casas-Solvas Juan M, Vargas-Berenguel Antonio, Barón Carmen, Ruiz-Pérez Luis M, González-Pacanowska Dolores, García-Fuentes Luis
Area de Química Física, Facultad de Ciencias Experimentales, Universidad de Almería, La Cañada de San Urbano, 04120 Almería, Spain.
Biochim Biophys Acta. 2008 Sep;1784(9):1347-55. doi: 10.1016/j.bbapap.2008.05.014. Epub 2008 Jun 9.
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).
脱氧尿苷5'-三磷酸核苷酸水解酶（dUTPase）催化dUTP水解生成dUMP和焦磷酸，在核苷酸代谢和DNA复制中发挥重要作用，可控制dTTP/dUTP的相对细胞水平，二者均可掺入DNA。等温滴定量热法已用于测定恶性疟原虫三聚体dUTPase的动力学和热力学参数，该酶是抗疟疾的潜在药物靶点。评估了二价离子在结合中的作用以及不同尿苷衍生物的抑制作用。当评估在EDTA存在下的dUTP水解时，与dUTP·Mg2+复合物相比，观察到k(cat)值降低了105倍，Km值增加了12倍。活化能E(a)和热力学活化参数的计算表明，当Mg2+耗尽时，能量屏障大约高4倍。其他二价离子如Co2+或Mn2+可以替代生理辅因子，然而与dUTP·Mg2+相比，k(cat)显著降低。通过ITC分析了dU、dUMP、dUDP和α,β-亚氨基-dUTP的结合和抑制作用，并与通过分光光度法和结合平衡研究获得的数据进行了比较。产物抑制（Kip dUMP：99.34 μM）不显著，但dUDP和α,β-亚氨基-dUTP的Ki值在低微摩尔范围内。还监测了离子强度对蛋白质稳定性的影响。DSC分析表明，随着盐浓度的增加，解折叠温度Tm略有升高。此外，在盐存在下的热解折叠途径充分符合不可逆的两态模型（N3→3D）。
