In this report, we demonstrated that the Yersinia protein tyrosine phosphatase (PTPase) could be inactivated by the alkylating agent iodoacetate. The enzyme modification was selective, and the covalent attachment was stoichiometric. The residue that was labeled by iodoacetate was shown to be Cys403, which was the same catalytically essential residue identified by site-directed utagenesis [Guan, K. L., & Dixon, J. E. (1990) Science 249, 553–556]. The rate of iodoacetate modification decreased as the ionic strength of the media increased. There was no significant D2O solvent isotope effect associated with the inactivation of the enzyme, suggesting that thiol anion of Cys403 reacted as a nucleophile. The Yersinia PTPase also displayed differential reactivity (940-fold) toward iodoacetate over iodoacetamide. This indicates that residues within the active site of the enzyme are positively charged. The pKa of the active site thiol group was determined to be 4.67. The low pKa value suggests that ionic interactions are important in stabilizing the thiolate anion. One candidate residue for this stabilization is the invariant histidine (His402) found in all PTPases. Substitutions of His402 with Asn or Ala altered the active site thiol pKa to 5.99 and 7.35, respectively. Interestingly, the active site thiol in the mutants also showed enhanced reactivity toward iodoacetate. The second-order rate constants for the inactivation of the wild-type enzyme, H402N, and H402A were 59.7, 3305, and 1763 M−1 min−1, respectively.
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