Active site labeling of a receptor-like protein tyrosine phosphatase.
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.
Pot
D A
DA
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Dixon
J E
JE
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D000814:Aniline Compounds; D006023:Glycoproteins; D007461:Iodoacetates; D009943:Organophosphorus Compounds; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C068798:4-aminophenylphosphate; D017027:Protein Tyrosine Phosphatases; C516845:Ptpra protein, rat; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D019807:Iodoacetic Acid