title: Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate.
abstract: 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 32P-labeled substrates and then rapidly denaturated, a 32P-labeled 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 32P-labeled p-nitrophenyl phosphate, 0.27 mol of phosphate was incorporated per mol of protein-tyrosine-phosphatase. 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 protein-tyrosine-phosphatase forms a covalent thiol phosphate linkage between Cys215 and phosphate.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: K L
authors.initials: KL
authors.lastname: Guan
authors.forename: J E
authors.initials: JE
authors.lastname: Dixon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases; D051381:Rats
chemical_list: D010750:Phosphoproteins; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine