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

The energetics of substrate dephosphorylation in the low-molecular-weight p rotein tyrosine phosphatase is studied with the empirical valence bond meth od in combination with molecular dynamics free energy perturbation simulati ons. Different mechanisms corresponding to different charge states of the r eacting groups are examined. We find very similar activation barriers for a ttack of the reactive cysteine anion on the mono- and dianion of phenylphos phate, although this reaction step is more exothermic in the latter case. T his result is found to be consistent with calculations of the relative bind ing affinities of the protonated and unprotonated substrate, which clearly indicate that the substrate dianion will not bind when the reactive cystein e 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 th at 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 cle avage. The calculated overall activation energy for substrate dephosphoryla tion according to the favored mechanism is in good agreement with experimen tal data. (C) 1999 John Wiley & Sons, Inc. Int J Quant Chem 73: 147-159, 19 99.