Mechanism of substrate dephosphorylation in low M-r protein tyrosine phosphatase

Substrate dephosphorylation by the low molecular weight protein tyrosine ph osphatases proceeds via nucleophilic substitution at the phosphorous atom y ielding a cysteinyl phosphate intermediate. However, several questions rega rding the exact reaction mechanism remain unanswered. Starting from the cry stal structure of the enzyme we study the energetics of this reaction, usin g the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The free energy profiles of two m echanisms corresponding to different protonation states of the reacting gro ups are examined along stepwise and concerted pathways. The activation barr iers calculated relative to the enzyme-substrate complex are very similar f or both monoanionic and dianionic substrates, but taking the substrate bind ing step into account shows that hydrolysis of monoanionic substrates is st rongly favored by the enzyme, because a dianionic substrate will not bind w hen the reacting cysteine is ionized. The calculated activation barrier for dephosphorylation of monoanionic phenyl phosphate according to this novel mechanism is 14 kcal mol(-1), which is in good agreement with experimental data. (C) 1999 Wiley-Liss, Inc.