Mechanism of substrate dephosphorylation in low Mr protein tyrosine phosphatase.
Substrate dephosphorylation by the low molecular weight protein tyrosine phosphatases proceeds via nucleophilic substitution at the phosphorous atom yielding a cysteinyl phosphate intermediate. However, several questions regarding the exact reaction mechanism remain unanswered. Starting from the crystal structure of the enzyme we study the energetics of this reaction, using the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The free energy profiles of two mechanisms corresponding to different protonation states of the reacting groups are examined along stepwise and concerted pathways. The activation barriers calculated relative to the enzyme-substrate complex are very similar for both monoanionic and dianionic substrates, but taking the substrate binding step into account shows that hydrolysis of monoanionic substrates is strongly favored by the enzyme, because a dianionic substrate will not bind when 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. Proteins 1999;36:370-379.
Department of Molecular Biology, Uppsala University, Biomedical Center, Uppsala, Sweden.
K
K
Kolmodin
P
P
Nordlund
J
J
Aqvist
D020134:Catalytic Domain; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008970:Molecular Weight; D010766:Phosphorylation; D011487:Protein Conformation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D017027:Protein Tyrosine Phosphatases