Thiol-Dependent Recovery of Catalytic Activity from Oxidized Protein Tyrosine Phosphatases

Abstract

Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme's catalytic cysteine thiolate group. Importantly, low molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively-inactivated PTPs (PTP1B and SHP-2) by various low molecular weight thiols and the enzyme thioredoxin. All thiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rates constants that varied by a factor of approximately eight. In general, molecules bearing low pKa thiol groups were particularly effective. The biological thiol, glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M−1 s−1, while the dithiol, DTT, displayed an apparent second-order rate constant of 0.325 ± 0.007 M−1 s−1. The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 μM) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10−3 s−1. The rates at which these agents regenerated oxidized PTP1B followed the trend Trx > DTT > GSH, with comparable values observed at 2 μM Trx, 4 mM DTT and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1-20 mM. The common biochemical reducing agent tris(2-carboxyethyl)phosphine (TCEP) regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M−1 s−1. We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events.

Redox Control of Microglial Function: Molecular Mechanisms and Functional Significance

“…Additional Trx targets include oxidized protein tyrosine phosphatases, for example, PTP1B. In certain cell signaling cascades, the production of H 2 O 2 inactivates PTP1B by oxidizing a cysteine sulfhydryl group (230). Trx has been shown to bind to apoptosis-signalregulating kinase 1 (ASK1), thereby blocking its activity and preventing both stress-and cytokine-mediated apoptosis.…”

Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM

“…1C), as was the PGP-catalyzed dephosphorylation of paranitrophenylphosphate (pNPP, Fig. 1D), a generic substrate that has been used to characterize redox-sensitive PTPs such as PTP-1B or SHP-2 [45]. due to a reversible, oxidative protein modification (Fig.…”

An increased need for dietary cysteine in support of glutathione synthesis may underlie the increased risk for mortality associated with low protein intake in the elderly

“…Glutathione opposes this effect, first, by functioning as a reducing substrate for glutathione peroxidase, which converts hydrogen peroxide to water (Arthur 2000). Second, via direct reactions with sulfenic acid, or via the catalytic impact of glutaredoxin, which likewise employs glutathione as a reducing substrate, glutathione can restore sulfenic acids to their native sulfhydryl form (Dickinson and Forman 2002;Shelton et al 2005;Parsons and Gates 2013). These protective mechanisms are evidently compromised when cellular levels of glutathione decline during the aging process.…”