| Abstract
| - The usual rate-determining step in the catalytic mechanism of the low molecular weight tyrosinephosphatases involves the hydrolysis of a phosphocysteine intermediate. To explain this hydrolysis, generalbase-catalyzed attack of water by the anion of a conserved aspartic acid has sometimes been invoked.However, experimental measurements of solvent deuterium kinetic isotope effects for this enzyme do notreveal a rate-limiting proton transfer accompanying dephosphorylation. Moreover, base activation of wateris difficult to reconcile with the known gas-phase proton affinities and solution phase pKa's of aspartic acidand water. Alternatively, hydrolysis could proceed by a direct nucleophilic attack by a water molecule. Tounderstand the hydrolysis mechanism, we have used high-level density functional methods of quantumchemistry combined with continuum electrostatics models of the protein and the solvent. Our calculationsdo not support a catalytic activation of water by the aspartate. Instead, they indicate that the water oxygendirectly attacks the phosphorus, with the aspartate residue acting as a H-bond acceptor. In the transitionstate, the water protons are still bound to the oxygen. Beyond the transition state, the barrier to protontransfer to the base is greatly diminished; the aspartate can abstract a proton only after the transitionstate, a result consistent with experimental solvent isotope effects for this enzyme and with establishedprecedents for phosphomonoester hydrolysis.
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