Phosphorothioates as models for studying phosphoryl transfer reactions

Phosphorothioates have long been used to study enzymatic phosphoryl transfer reactions. While the reactions in solution of phosphate monoesters have been well characterized, less is known about their analogs, phosphorothioates. This work further investigates if phosphorothioate monoesters are proper models to study enzymatic phosphoryl transfer reactions. The results obtained for reactions of para-nitrophenyl phosphorothioate in solution, as well as enzymatic reactions, are compared to the data reported over the years for the corresponding reactions of para-nitrophenyl phosphates. The mechanism of the reaction in solution was studied using kinetic and thermodynamic tools. Insights regarding the transition state for the reaction in solution of para-nitrophenyl phosphorothioates were gained from kinetic isotope effects. The enzymatic reactions catalyzed by alkaline phosphatase from Escherichia coli and Yersinia protein phosphatase of para-nitrophenyl phosphorothioate were also investigated. The kinetic isotope effects were compared with the available data for para-nitrophenyl phosphate. The equilibrium isotope effects determined for the protonation of para-nitrophenyl phosphorothioate, and metal complexation of para-nitrophenyl phosphorothioate dianion helped us to interpret the kinetic isotope effects measured for the enzymatic reactions. Linear free energy relationship studies were employed to determine the reliability of thio effects in inferring mechanistic conclusions for enzymatic reactions of phosphates. Computational studies were performed to investigate the differences in the binding of phenyl phosphate and phenyl phosphorothioates in the active site of alkaline phosphatase from Escherichia coli. Kinetic isotope effects made it possible to compare two phosphatases from two different classes: alkaline phosphatase from Escherichia coli, which uses a binuclear metal center, and Yersinia protein phosphatase, which does not utilize metal ions and uses a general acid/general base type of mechanism.