Kinetic properties and specificity of trimeric Plasmodium falciparum and human dUTPases.
Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. Hydrolysis of other nucleotides similar in structure to dUTP would be physiologically negative and therefore high substrate specificity is essential. Binding and hydrolysis of nucleotides different to dUTP by the dUTPases from Plasmodium falciparum (PfdUTPase) and human (hdUTPase) was evaluated by applying isothermal titration calorimetry (ITC). The ribo and deoxyribonucleoside triphosphates dGTP, dATP, dCTP, dTTP, UTP, FdUTP and IdUTP have been analysed. dUTP and FdUTP were the most specific substrates for both enzymes. The specificity constants (k(cat)/K(m)) for the remaining ones, except for the IdUTP, were very similar for both enzymes, although PfdUTPase showed a slightly higher specificity for dCTP and UTP and the human enzyme for dTTP and dCTP. PfdUTPase was very efficient in using FdUTP as substrate indicating that small size substituents in the 5' position are well tolerated. In addition product inhibition was assessed by binding studies with the nucleoside monophosphate derivatives and thermodynamic parameters were established. When FdUTP hydrolysis was monitored, Plasmodium dUTPase was more sensitive to end-product inhibition by FdUMP than the human enzyme. Taken together these results highlight further significant differences between the human and Plasmodium enzymes that may be exploitable in selective inhibitor design.
Quesada-Soriano
Indalecio
I
Universidad de Almería, La Cañada de San Urbano, Spain.
Casas-Solvas
Juan M
JM
Recio
Eliseo
E
Ruiz-Pérez
Luis M
LM
Vargas-Berenguel
Antonio
A
González-Pacanowska
Dolores
D
García-Fuentes
Luis
L
D002151:Calorimetry; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D010963:Plasmodium falciparum / Q000201:enzymology*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D003856:Deoxyuracil Nucleotides; D004791:Enzyme Inhibitors; C027078:deoxyuridine triphosphate; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase