Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate.

摘要: 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 react with 32P-labeled substrates then rapidly denaturated, phosphoprotein could be visualized electrophoresis. Transient formation of observed, protein disappeared as substrate consumed. In presence p-nitrophenyl phosphate, 0.27 mol phosphate incorporated per protein-tyrosine-phosphatase. Site-directed mutagenesis catalytically essential cystine residue (position 215) resulted an inactive enzyme, no formed. The showed maximum lability between pH 2.5 3.5 decomposed iodine. These properties, along additional site-directed mutations, suggest that forms covalent thiol linkage Cys215 phosphate.