Calorimetric determination of thermodynamic parameters of 2′-dUMP binding to Leishmania major dUTPase

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 degreesC, 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 DeltaGdegrees remains practically invariant as a function of temperature, both DeltaH and DeltaSdegrees decrease with increasing temperature. The T-S and T-H were 23.4 and 13.6 degreesC, respectively. The temperature dependence of the enthalpy change yields a heat capacity change of DeltaC(p)degrees = -618.1 +/- 126.4 cal (.) mol(-1) (.) 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. (C) 2004 Elsevier B.V. All rights reserved.