Pot D A, Dixon J E
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1992 Jan 5;267(1):140-3.
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.
碘乙酸而非碘乙酰胺可使大鼠LAR（一种受体样蛋白酪氨酸磷酸酶，PTPase）的胞质结构域失活，这表明碘乙酸以高度选择性的方式与该酶相互作用。数据表明，碘乙酸以每摩尔大鼠LAR结合0.8摩尔碘乙酸的化学计量比结合在该酶的活性位点。在用内肽酶Lys-C消化放射性标记的PTPase后，分离出了一个单一的[14C]碘乙酸标记的肽段。对活性位点标记肽段的序列分析表明，Cys-1522含有放射性标记。定点诱变已表明该残基对大鼠LAR活性至关重要（Pot, D. A., Woodford, T. A., Remboutsika, E., Haun, R. S., and Dixon, J. E. (1991) J. Biol. Chem. 266, 19688 - 19696）。碘乙酸仅与这种双结构域PTPase的第一个结构域发生反应。这些结果首次直接鉴定出了PTPase活性位点处高反应性的半胱氨酸残基，并突出了该残基作为亲核试剂参与从酪氨酸水解磷酸的能力。
