ENERGETICS OF NUCLEOPHILE ACTIVATION IN A PROTEIN-TYROSINE-PHOSPHATASE

The nucleophilic attack by cysteine 12 in the low-molecular-weight pro tein tyrosine phosphatase is believed to be carried out by the thiolat e anion form of this residue. We here study the energetics of proton t ransfer between the thiol group of cysteine 12 and a substrate phospha te oxygen atom, to examine the effects of the enzymic environment on t he stability of the thiolate nucleophile. This is done by molecular dy namics and free energy perturbation simulations, utilizing the empiric al valence bond method to describe the potential surface of the system . The calculations show that the protein environment significantly sta bilizes the thiolate ion, thereby setting the stage for the nucleophil ic attack. We compare these results with those from further simulation s of a mutant enzyme, and demonstrate the importance of serine 19 in t hiolate stabilization. (C) 1997 Academic Press Limited