The catalytic role of Cys124 in the dual specificity phosphatase VHR.
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.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
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G
Zhou
J M
JM
Denu
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Wu
J E
JE
Dixon
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D003545:Cysteine; D017931:DNA Primers; D054640:Dual Specificity Phosphatase 3; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006801:Humans; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014616:Vaccinia virus
D017931:DNA Primers; D007461:Iodoacetates; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517025:DUSP3 protein, human; D054640:Dual Specificity Phosphatase 3; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine; D019807:Iodoacetic Acid