Computational modeling of the rate limiting step in low molecular weight protein tyrosine phosphatase

Hydrolysis of the phosphoenzyme intermediate is the second and rate limitin g step of the reaction catalyzed by the protein tyrosine phosphatases (PTPs ), The cysteinyl phosphate thioester bond is cleaved by nucleophilic displa cement where an active site water molecule attacks the phosphorus atom. Sta rting from the crystal structure of the low molecular weight PTP, we study the energetics of this reaction utilizing the empirical valence bond method in combination with molecular dynamics and free energy perturbation simula tions. The reactions of the wild-type as well as the D129A and C17S mutants are modeled. For the D129A mutant, which lacks the general acid/base resid ue Asp-129, an alternative reaction mechanism is proposed. The calculated a ctivation barriers are in all cases in good agreement with experimental rea ction rates. The present results together with earlier computational and ex perimental work now provide a detailed picture of the complete reaction mec hanism in many PTPs, The key role played by the structurally invariant sign ature motif in stabilizing a double negative charge is reflected by its con trol of the energetics of both transition states and the reaction intermedi ate. (C) 1999 Federation of European Biochemical Societies.