Energetics of nucleophile activation in a protein tyrosine phosphatase

The nucleophilic attack by cysteine 12 in the low-molecular-weight protein tyrosine phosphatase is believed to be carried out by the thiolate anion form of this residue. We here study the energetics of proton transfer between the thiol group of cysteine 12 and a substrate phosphate oxygen atom, to examine the effects of the enzymic environment on the stability of the thiolate nucleophile. This is done by molecular dynamics and free energy perturbation simulations, utilizing the empirical valence bond method to describe the potential surface of the system. The calculations show that the protein environment significantly stabilizes the thiolate ion, thereby setting the stage for the nucleophilic attack. We compare these results with those from further simulations of a mutant enzyme, and demonstrate the importance of serine 19 in thiolate stabilization.