Role of water in the energy of hydrolysis of phosphoanhydride and phosphoester bonds

The observed equilibrium constants for hydrolysis (Kobs) of a phosphoester and a phosphoanhydride bond were measured under a variety of conditions likely to alter the interactions of reactants and products with water. These included increasing the pH of the medium from 5.0 to 10.0, increasing the MgCl2 concentration form 0 to 200 mM, and decreasing the water activity of the medium by adding either dimethyl sulfoxide (50%, v/v) or polyethylene glycol 6,000-8,000 (50%, w/v). The Kobs for phosphoesters such as phosphoserine, glucose phosphate, glycerol phosphate, and ethylene glycol phosphate varied little over this wide range of conditions, the extreme values of Kobs being 12 and 200 M. In contrast, the Kobs for the phosphoanhydride bond of pyrophosphate varied from a value greater than 20,000 to 0.1 M. In totally aqueous media at a pH between 7.0 and 8.0 and in the presence of 0.5-1.0 mM MgCl2, the energy of hydrolysis of pyrophosphate was 1.2-4.0 kcal/mol greater than that of phosphoserine. However, when the water activity was decreased by adding polyethylene glycol to the medium within the same pH and MgCl2 concentration range, the energy of hydrolysis of phosphoserine became 2.0-2.5 kcal/mol greater than that of pyrophosphate. The results suggest that for phosphoesters, the solvation energies of reactants and products, unlike the case of phosphoanhydride bonds, are not the major factors in determining the energy of hydrolysis.