Computational modeling of catalysis and binding in low-molecular-weight protein tyrosine phosphatase

The energetics of substrate dephosphorylation in the low-molecular-weight protein tyrosine phosphatase is studied with the empirical valence bond method in combination with molecular dynamics free energy perturbation simulations. Different mechanisms corresponding to different charge states of the reacting groups are examined. We find very similar activation barriers for attack of the reactive cysteine anion on the mono- and dianion of phenylphosphate, although this reaction step is more exothermic in the latter case. This result is found to be consistent with calculations of the relative binding affinities of the protonated and unprotonated substrate, which clearly indicate that the substrate dianion will not bind when the reactive cysteine is in its thiolate form. The reaction with monoanionic substrate is found to have an activation barrier that is more than 15 kcal/mole lower than that of the dianion when the binding step is taken into account. We also find that leaving group protonation by Asp129 has to be concerted with bond cleavage. The calculated overall activation energy for substrate dephosphorylation according to the favored mechanism is in good agreement with experimental data.