The PTPs employ covalent catalysis, utilizing the thiol group of the active-site Cys residue as the attacking nucle-ophile to form a thiophosphoryl enzyme intermediate (E-P) . Substitutions of the Cys residue completely abrogate PTP activity. The nucleophilic cysteine is housed within the active-site architecture specifically designed to bind a negatively charged phosphoryl group. Consequently, the pKa for the sulfhydryl group of the active-site Cys is extremely low (~ 5) . Thus, the PTPs are very sensitive to thiol-specific alkylating agents. For example, the PTPs can be irreversibly inactivated by iodoacetate, N-ethylmaleimide, and 5,5'-dithio-2-nitrobenzoic acid [7-9]. In addition, PTPs can also be inactivated by heavy metals including Zn2+, Cu2+, and p-(hydroxymercuri)benzoate, possibly through covalent bond formation with the active-site thiol group. There were
several attempts to design specific, PTP-active-site-directed, alkylating agents, taking into consideration the architecture of the PTP catalytic site and the nature of the thiol-mediated phosphate hydrolysis. The 4-fluoromethylphenylphosphate (Fig. 1, compound 1) was designed as a mechanism-based phosphatase inactivator [10,11] that, upon cleavage of the phosphate ester bond by the phosphatase, rapidly liberates the fluoride ion and forms a reactive quinone methide intermediate. Subsequent attack by PTP nucleophilic residues would result in formation of covalent adducts. Unfortunately, the lack of selectivity among various phosphatases and the unfavorable kinetics prevent the wide use of this compound in PTP research. The a-halobenzylphosphonate (Fig. 1, compound 2) is an irreversible inactivator of the Yersinia PTP and PTP1B . Mechanistically, this compound would be expected to undergo nucleophilic displacement of the halide without cleavage of the carbon-phosphorus bond. More recently, a-haloacetophenone derivatives (Fig. 1, compound 3) have also been shown to be capable of cova-lently modifying SHP1 and PTP1B, possibly via nucleophilic displacement of the halide by the active-site thiol group . A unique feature of these compounds is that the inhibition can be reversed upon photoactivation. Interestingly, the dipeptide aldehyde calpain inhibitor Calpeptin (Fig. 1, compound 4) was recently shown to preferentially inhibit membrane-associated PTPs . Although the exact mechanism of inhibition has not been investigated, it is possible that the aldehyde functionality may react with residues in the PTP active site to form covalent adducts. Finally, several vitamin K analogs (Fig. 1, compound 5) have been shown to be effective PTP inactivators, possibly involving Michaeltype nucleophilic addition of the active site Cys to the mena-dione moiety .
Due to the extremely low pKa of the active-site thiol group, the PTPs are also prone to metal ion-catalyzed oxidation by O2 in the air. Thus, it is a common practice to include EDTA and DTT in PTP assay buffers in order to keep the active-site Cys in the reduced form. In addition to molecular oxygen, exposure of the PTPs to reactive oxygen species (ROS) can also result in PTP inactivation. For example, it has been shown that treatment of various PTPs with hydrogen peroxide [16,17], superoxide radical anion , and nitric oxide  all lead to the oxidation of the active-site Cys. Because ROS can be generated endogenously in the cell and because the oxidation of the active-site Cys by ROS in many cases is reversible, it has been suggested that PTP inactivation by ROS may provide a means for temporal negative regulation of PTP activity.
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