The thermodynamics, pH dependency, solvent effects, and the effect of divalent metal ions have been examined for the hydrolysis reactions of the phosphorothioate monoester-p-nitrophenyl phosphorothioate (pNPPT) and compared with those of the corresponding phosphate monoester, p-nitrophenyl phosphate (pNPP). The pH dependency for pNPPT hydrolysis mirrors that for typical phosphate monoesters, with the monoanion being the most reactive species. The ratio of the rate constants for pNPPT hydrolysis to that for pNPP is 1380 for the monoanion reactions and 12.6 for the dianion reactions at 39 degrees C. The free energy of activation for hydrolysis of the pNPPT dianion in water is 27.9 kcal/mol versus 29.5 kcal/mol for pNPP; for the monoanion reactions the values are 22.2 kcal/mol for pNPPT and 26.8 kcal/mol for pNPP. The free energies of solvation for pNPPT and pNPP are within 0.1 kcal/mol of each other despite the poorer hydrogen bonding ability of sulfur versus oxygen. This minimal difference in ground-state energies thus does not account for the difference in activation energies, which must therefore arise from transition state effects. The more favorable Delta G(double dagger) for pNPPT dianion hydrolysis versus pNPP is entropic in origin; the enthalpic barrier is greater in the pNPPT reaction but is more than offset by a more favorable entropy of activation that arises from the fully dissociative mechanism followed by pNPPT. By contrast, the more favorable Delta G(double dagger) for hydrolysis of the pNPPT monoanion compared to the pNPP monoanion is enthalpic in origin. The hydrolysis rate of the pNPPT dianion increases by nearly 10(7)-fold as DMSO content is changed from 0 to 95%. The rate acceleration is due to a lower enthalpy of activation in the mixed solvent. In contrast, the rate of hydrolysis of the monoanion of pNPPT in aqueous DMSO remains almost unchanged from the aqueous value; the enthalpic barrier in 95% DMSO is decreased but is offset by an increase in the entropic barrier. The kinetic effects on hydrolysis of the pNPPT dianion caused by complexation with magnesium, which coordinates with oxygen in phosphorothioates, and cadmium, which coordinates with sulfur, were found to be similar with each metal, resulting in a small decrease in the rate constant.