We have investigated the binding of 2'-deoxyuridine 5'-monophosphate (2'-dUMP) to Leishmania major deoxyuridine 5'-triphosphate nucleotide hydrolase (dUTPase) by isothermal titration microcalorimetry under different experimental conditions. Binding to dimeric L. major dUTPase is a non-cooperative process, with a stoichiometry of 1 molecule of 2'-dUMP per subunit. The utilization of buffers with different ionization enthalpies has allowed us to conclude that the formation of the 2'-dUMP-dUTPase complex, at pH 7.5 and 30 degrees C, is accompanied by the uptake of 0.33 +/- 0.05 protons per dUTPase subunit from the buffer media. Moreover, 2'-dUMP shows a moderate affinity for the enzyme, and binding is enthalpically driven across the temperature range studied. Besides, whereas DeltaG degrees remains practically invariant as a function of temperature, both DeltaH and DeltaS degrees decrease with increasing temperature. The TS and TH were 23.4 and 13.6 degrees C, respectively. The temperature dependence of the enthalpy change yields a heat capacity change of DeltaCp degrees = -618.1 +/- 126.4 cal x mol(-1) x K(-1), a value low enough to discard major conformational changes, in agreement with the fitting model. An interpretation of this value in terms of solvent-accessible surface areas is provided.