Microcalorimetry, high-performance liquid chromatography (h.p.l.c.), and in enzymatic assay have been used to conduct a thermodynamic investigation of five phosphate hydrolysis reactions: {alpha-D-galactose 1-phosphate(aq)+H2O(1) = D-galactose(aq) + orthophosphate(aq)} (1), {sn-glycerol 3-phosphate(aq) + H2O(1)=glycerol(aq) + orthophosphate(aq)} (2). {4-nitrophenyl phosphate(aq) + H2O(1) 4-nitrophenol(aq) + orthophosphate(aq)} (3), {phosphocreatine(aq) + H2O(1) = creatine(aq) + orthophosphate(aq)} (4), and {3-phospho=D-glycerate(aq) + H2O (1) =D-glycerate(aq) + orthophosphate(aq)} (5). Calorimetrically determined enthalpies of reaction Delta H-r(cal) were measured for reactions (1)-(5) and the apparent equilibrium constant K' was measured for reaction (2). The pKs and standard enthalpies of reaction Delta H-r for the H+ and Mg2+ binding reactions of the reactants and products in the aforementioned reactions were obtained either from the literature or by estimation. A chemical equilibrium model was then used to calculate standard equilibrium Constants K and standard enthalpies of reaction Delta H-r for chemical reference reactions that correspond to the overall biochemical reactions that Were Studied experimentally. Property Values from the literature and thermodynamic network Calculations were used to obtain values of the equilibrium constants for the chemical reference reactions that correspond to the overall biochemical reactions (1). These values were compared with other results from the literature and also correlated With Structural features. The results obtained in this Study can be used in the chemical equilibrium model to calculate values of K', the standard apparent Gibbs free energy changes Delta(r)G', the standard apparent enthalpy changes Delta H-r", changes in binding of the proton Delta N-r(H+), and the position of equilibrium for the overall biochemical reactions considered in this study over a reasonably wide range of temperature. pH, pMg [.. log(10){m(Mg2+)/m degrees}], and ionic strength I. Values of K' and Delta(r)G'degrees under approximately physiological conditions (T = 310.15 K, pH 7.0, pMg = 3.0, and I-m = 0.25 mol . kg(-1)) have been calculated. (C) 2008 Elsevier Ltd. All rights reserved.