Evidence for Protein - tyrosine - phosphatase Cysteine - Phosphate Intermediate

A recombinant protein-tyrosine-phosphatase has been expressed in Escherichia coli and purified to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using affinity chromatography. When the phosphatase was allowed to react with 32Plabeled substrates and then rapidly denaturated, a 32Plabeled phosphoprotein could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transient formation of a 32P-labeled phosphoprotein was observed, and the 32P-labeled protein disappeared as substrate was consumed. In the presence of ”Plabeled p-nitrophenyl phosphate, 0.27 mol of phosphate was incorporated per mol of protein-tyrosinephosphatase. Site-directed mutagenesis of a catalytically essential cystine residue (position 215) in the recombinant protein resulted in an inactive enzyme, and no phosphoprotein was formed. The 32P-labeled phosphoprotein showed a maximum lability between pH 2.5 and 3.5 and was rapidly decomposed in the presence of iodine. These properties, along with additional site-directed mutations, suggest that the proteintyrosine-phosphatase forms a covalent thiol phosphate linkage between CysZ1‘ and phosphate.