Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase

Abstract

Covalent modification experiments were conducted in order to identify active site residues of the 18-kDa cytoplasmic phosphotyrosyl protein phosphatases. The enzyme was inactivated by diethyl pyrocarbonate, phenylglyoxal, cyclohexanedione, iodoacetate, iodoacetamide, phenylarsine oxide, and certain epoxides in a manner consistent with the modification of active site residues. Phenylglyoxal and cyclohexanedione both bind to the active site in a rapid preequilibrium process and thus act as active site-directed inhibitors. The pH dependencies of the inactivation by iodoacetate and by iodoacetamide were examined in detail and compared with rate data for the alkylation of glutathione as a model compound. The enzyme inactivation data permitted the determination of pKa values of two reactive cysteines at or near the active site. Although phosphomycin is simply a competitive inhibitor of the enzyme, it was found that 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and (R)- and (S)-benzylglycidol act as irreversible covalent inactivators, consistent with the importance of a hydrophobic moiety on the substrate in controlling substrate specificity. EPNP exhibits characteristics of an active site-directed inactivator, with a preequilibrium binding constant somewhat smaller than that of phosphate ion. The pH dependencies of inactivation of EPNP and (S)-benzylglycidol are identical to that observed for iodoacetamide and similar to that for iodoacetate, suggesting that they modify similar groups. Sequencing of the tryptic digests of the EPNP-labeled enzyme indicates that Cys-62 and Cys-145 are labeled. Phenylarsine oxide acts as a very slow, tight-binding inhibitor of the enzyme. The results are interpreted in terms of an active site model that incorporates a histidine-cysteine ion pair, similar to that present in papain.

Advanced Glycation End Product Precursors Impair Epidermal Growth Factor Receptor Signaling

“…Dicarbonyls may induce nonenzymatic modifications of various amino acids (Cys, Lys, or Arg) that are generally present in the putative active site of the receptor tyrosine kinase family (31). Some other hypothetical mechanisms potentially involved in dicarbonyl-induced EGFR inhibition have been excluded: 1) GO and MGO were not cytotoxic to cultured cells under the conditions used in the experiment; 2) the loss of EGFR activity was not associated with a loss of EGFR protein but rather resulted from a reduced specific activity of the EGFR kinase; 3) the GO/MGO-induced inhibition of EGFR phosphorylation does not result from activation of PTPases (involved in EGFR dephosphorylation) because, under our experimental conditions, GO/MGO strongly inhibited PTPases, in agreement with a previous report (32); and 4) GO/MGOinduced EGFR inhibition is not prevented by antioxidants such as ␣-tocopherol and N-acetyl-cysteine (data not shown).…”

Methylglyoxal Impairs the Insulin Signaling Pathways Independently of the Formation of Intracellular Reactive Oxygen Species

“…For the deleterious action of methylglyoxal on the insulin signaling pathway, the decrease in tyrosine-phosphorylated IRS proteins was not due to impaired IRS synthesis/degradation as, after this short methylglyoxal treatment, IRS-1 protein levels were unchanged. Further, the reduction in IRS-1 tyrosine phosphorylation is not likely to be due to increased tyrosine dephosphorylation, as previous studies have reported a strong inhibition of phosphotyrosine phosphatases by ␣-ketoaldehydes (29,47). The decrease in insulin-stimulated phosphorylation of IRS-1 and IRS-2 (data not shown) on tyrosine was associated with a reduction in p85-IRS-1 association, PI3K activity, PKB activation, and hormone stimulation of glucose transport.…”

Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

“…In fact, the reduced state of all the sulfhydryl groups of LMW-PTP has been demonstrated by our group and others (25,26). Both cysteines in positions 12 and 17 are conserved among the LMW-PTP subfamily, thus suggesting an important role for both residues.…”

“…We have already demonstrated that Cys-12 is the essential cysteine residue performing in the cysteinyl-phosphate intermediate during catalysis, whereas Cys-17 has a role in phosphate binding in cooperation with Arg-18 (27). Van Etten et al (26) have demonstrated that LMW-PTP has two reactive cysteines in the catalytic site with a low pK a (at pH 7.52 and 9.05 for iodoacetamide). Caselli et al (11,12) have demonstrated that H 2 O 2 and NO lead to the specific oxidation of Cys-12 and Cys-17 in the catalytic pocket of LMW-PTP.…”

“…Caselli et al (11,12) have demonstrated that H 2 O 2 and NO lead to the specific oxidation of Cys-12 and Cys-17 in the catalytic pocket of LMW-PTP. In addition the active site cysteines of LMW-PTP, as in other PTPs, are specifically targeted by the sulfhydryl-modifying reagent iodoacetic acid (25,26). Recently an iodoacetamide-fluorescein (5Ј-F-IAA) labeling method of proteins containing low pK a cysteine residues has been reported (28), and we applied this method to study the oxidation state of LMW-PTP in vivo.…”