Zhou G, Denu J M, Wu L, Dixon J E
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
J Biol Chem. 1994 Nov 11;269(45):28084-90.
The recombinant human Vaccinia virus H1-related protein tyrosine phosphatase, (VHR PTPase) possesses intrinsic Tyr and Thr/Ser phosphatase activities. Both activities were abolished by a single amino acid substitution, C124S. When VHR was incubated with a 32P-labeled phosphotyrosine-containing substrate and then rapidly denatured, enzyme-associated 32P was evident following SDS-polyacrylamide gel electrophoresis. The formation of 32P-labeled protein could be blocked in the presence of an unlabeled substrate. VHR-associated 32P was sensitive to iodine but insensitive to pyridine and hydroxylamine. The catalytically inactive C124S mutant would not form a 32P-labeled enzyme. Furthermore, VHR phosphatase could be selectively inactivated by the alkylating agent iodoacetate. The inactivation resulted from the specific covalent modification of Cys124. Collectively these results suggest that a thiol-phosphate enzyme intermediate is formed when Cys124 of VHR accepts a phosphate from the substrate. Our results also demonstrate that the dual specificity phosphatases and the tyrosine-specific PTPases employ similar catalytic mechanisms.
重组人痘苗病毒H1相关蛋白酪氨酸磷酸酶（VHR PTPase）具有内在的酪氨酸和苏氨酸/丝氨酸磷酸酶活性。这两种活性都因单个氨基酸取代C124S而丧失。当VHR与32P标记的含磷酸酪氨酸底物一起孵育，然后迅速变性时，在SDS-聚丙烯酰胺凝胶电泳后可明显看到与酶结合的32P。在未标记底物存在的情况下，32P标记蛋白的形成会被阻断。与VHR相关的32P对碘敏感，但对吡啶和羟胺不敏感。催化无活性的C124S突变体不会形成32P标记的酶。此外，VHR磷酸酶可被烷基化剂碘乙酸选择性灭活。这种失活是由Cys124的特异性共价修饰导致的。这些结果共同表明，当VHR的Cys124从底物接受一个磷酸基团时，会形成硫醇-磷酸酶中间产物。我们的结果还表明，双特异性磷酸酶和酪氨酸特异性PTP酶采用相似的催化机制。
