Protein tyrosine phosphatases (PTPs) regulate important biochemical processes by catalysing the hydrolysis of phosphotyrosine from other proteins. Two members from this family are the dual specificity phosphatases VHR and CDC25B. The first step of catalysis is the nucleophilic attack from a PTP cysteine side chain toward the phosphorus substrate, with a possible H+ transfer from a general acid to the leaving group. A thiophosphorylated PTP intermediate is formed and the substrate is dephosphorylated. Uncertainties about this step involve: the protonation states of substrate and enzyme nucleophile; the inactivity of certain mutants; and the identification of the general acid in CDC25s.
These questions were investigated by computer simulations and determination of free-energy profiles for reactions of different substrates catalysed by VHR and CDC25B wild-types and mutants. A hybrid QM/MM potential was used. This potential was fully tested and calibrated, employing ab initio data on phosphate ester model reactions. Results show the catalysed reaction mechanism follows a concerted addition and elimination, with a dissociative transition state. Calculated barriers are very close to experimental activation energies. The substrate is a deprotonated dianion and the nucleophile is ionised. Reactions of the protonated substrate or nucleophile have barriers at least 15 kcal/mol higher than the experimental results. Active site cysteine to serine VHR mutants are inactive because the serine is protonated. CDC25s do not employ a general acid for catalysis, differently from other PTPs. Proposals that the general acid is the substrate or one of the glutamic acids present in the active site are not energetically accessible.