Plasmodium falciparum dUTPase: studies on protein stability and binding of deoxyuridine derivatives.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme preventing a deleterious incorporation of uracil into DNA, has been thought of as a novel target for anticancer and antiviral drug design. The interaction of Plasmodium falciparum dUTPase (PfdUTPase) with deoxyuridine derivatives (dU, dUMP, dUDP and dUpNHpp) has been studied thermodynamically by both isothermal titration and differential scanning calorimetry. ITC shows no cooperativity for the binding of these derivatives. Dependencies in the binding thermodynamic parameters (enthalpy, entropy and Gibbs energy changes) with the number of phosphate groups in the nucleotide are obtained, and from the heat capacity changes no significant conformational changes upon binding are inferred. DSC shows PfdUTPase trimer is very stable but denatures irreversibly, with a more complex denaturation profile than other homologous trimeric dUTPases. The presence of magnesium ions does not influence the denaturation profile, while the presence of deoxyuridine derivatives increases the stability. The increase depends upon nucleotide concentration and type, with dUDP having the greater effect.

Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase.

dUTPase (deoxyuridine 5'-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5'-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k(cat) and a 15-fold increase in the K(m) for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mg(2+) independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.