Structural and mechanistic basis for the activation of a low-molecular weight protein tyrosine phosphatase by adenine

Although the activation of low-molecular weight protein tyrosine phosphatas es by certain purines and purine derivatives was first described three deca des ago, the mechanism of this rate enhancement was unknown. As an example, adenine activates the yeast low-molecular weight protein tyrosine phosphat ase LTP1 more than 30-fold. To examine the structural and mechanistic basis of this phenomenon, we have determined the crystal structure of yeast LTP1 complexed with adenine, In the crystal structure, an adenine molecule is f ound bound in the active site cavity, sandwiched between the side chains of two large hydrophobic residues at the active site. Hydrogen bonding to the side chains of other active site residues, as well as some water-mediated hydrogen bonds, also helps to fix the position of the bound adenine molecul e. An ordered water was found in proximity to the bound phosphate ion prese nt in the active site, held by hydrogen bonding to N3 of adenine and O delt a 1 of Asp-132. On the basis of the crystal structure, we propose that this water molecule is the nucleophile that participates in the dephosphorylati on of the phosphoenzyme intermediate. Solvent isotope effect studies show t hat there is no rate-determining transfer of a solvent-derived proton in th e transition state for the dephosphorylation of the phosphoenzyme intermedi ate. Such an absence of general base catalysis of water attack is consisten t with the stability of the leaving group, namely, the thiolate anion of Cy s-13. Consequently, adenine activates the enzyme by binding and orienting a water nucleophile in proximity to the phosphoryl group of the phosphoenzym e intermediate, thus increasing the rate of the dephosphorylation step, a s tep that is normally the rate-limiting step of this enzymatic reaction.