Active site labeling of a receptor-like protein tyrosine phosphatase

Abstract

The inactivation of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase (PTPase), by iodoacetate and not by iodoacetamide suggested that iodoacetate interacts in a highly selective manner with the enzyme. The data indicate that iodoacetate binds at the active site of the enzyme with a stoichiometry of 0.8 mol of iodoacetate bound per mol of rat LAR. A single [14C]iodoacetate-labeled peptide was isolated following endoproteinase Lys-C digestion of the radiolabeled PTPase. Sequence analysis of the active site labeled peptide demonstrates that Cys-1522 contains the radiolabel. This residue has been shown by site-directed mutagenesis to be essential for rat LAR activity (Pot, D. A., Woodford, T. A., Remboutsika, E., Haun, R. S., and Dixon, J. E. (1991) J. Biol. Chem. 266, 19688-19696). Iodoacetate reacts only with the first domain of this double domain PTPase. These results, for the first time, directly identify the highly reactive cysteine residue at the active site of a PTPase and highlight the ability of this residue to participate as a nucleophile in the hydrolysis of phosphate from tyrosine.