THE CATALYTIC ROLE OF CYS(124) IN THE DUAL-SPECIFICITY PHOSPHATASE VHR

The recombinant human Vaccinia virus H1-related protein tyrosine phosp hatase, (VHR PTPase) possesses intrinsic Tyr and Thr/Ser phosphatase a ctivities. Both activities were abolished by a single amino acid subst itution, C124S. When VHR was incubated with a P-32-labeled phosphotyro sine-containing substrate and then rapidly denatured, enzyme-associate d P-32 was evident following SDS-polyacrylamide gel electrophoresis. T he formation of P-32-labeled protein could be blocked in the presence of an unlabeled substrate. VHR-associated P-32 was sensitive to iodine but insensitive to pyridine and hydroxylamine. The catalytically inac tive C124S mutant would not form a P-32-labeled enzyme. Furthermore, V BR phosphatase could be selectively inactivated by the alkylating agen t iodoacetate. The inactivation resulted from the specific covalent mo dification of Cys(124). Collectively these results suggest that a thio l-phosphate enzyme intermediate is formed when Cys(124) of VHR accepts a phosphate from the substrate. Our results also demonstrate that the dual specificity phosphatases and the tyrosine-specific PTPases emplo y similar catalytic mechanisms.