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 monothiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rate constants that varied by a factor of approximately 8. 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 dithiothreitol (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 order Trx > DTT > GSHand 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 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.

Figure 1

Thiol-mediated reactivation of oxidatively inactivated PTP1B. (A) PTP1B_ox (22 nM) was incubated with various thiols in Buffer A containing the chromogenic PTP substrate pNPP (20 mM), pH 7 at room temperature. The time courses monitored the increase in absorbance at 410 nm resulting from the PTP1B-catalyzed release of p-nitrophenol from the substrate. Monothiols were 50 mM and dithiols 25 mM: TGA-OMe (*), DTT (), BAL (+), TGA (), 2 ME (), GSH (), NAC (-), no thiol (). (B) Instantaneous rates of reactivation (slopes) versus time. Data were fit with pseudo-first order kinetic treatment to give the following estimates of the apparent bimolecular rate constants calculated in units of M⁻¹ s⁻¹: TGA-OMe = 0.45, DTT = 0.33, BAL = 0.33, TGA = 0.12, 2-ME = 0.08, GSH = 0.04, NAC = 0.04.

Figure 2

Reactivation of oxidatively-inactivated PTP1B by glutathione (GSH) and DTT. (A) GSH-mediated recovery of activity from oxidized PTP1B. Concentrations of GSH were 10, 20, 30, 40, and 50 mM (bottom to top). (B) Pseudo-first order rate constants (s⁻¹ × 10³) were plotted against corresponding concentrations of GSH (mM), affording a straight line of slope k_obs (M⁻¹ s⁻¹) which passes through the origin. (C) Time-course for DTT-mediated recovery of activity from oxidized PTP1B. Concentrations of DTT were 2.5, 5, 10, 15, and 20 mM (bottom to top). (D) Pseudo-first order rate constants (s⁻¹ × 10³) were plotted against corresponding concentrations of DTT (mM), affording a straight line of slope k_obs (M⁻¹ s⁻¹) which passes through the origin. A higher concentration regime of DTT (40-60 mM) was also explored in separate experiments conducted under identical conditions. No saturation behavior was observed under any of these concentration regimes.

Figure 3

Reactivation of oxidatively-inactivated PTP1B by DTT or the thioredoxin-thioredoxin reductase system. To solutions of 2×-concentrated reducing system in Buffer R were added 1:1 (v/v) oxidatively-inactivated PTP1B to final concentrations: PTP1B_ox (350 nM), DTT (5 mM, blue circles) or thioredoxin/thioredoxin reductase/NADPH (2 μM, 100 nM, 200 μM, respectively, red triangles). Immediately upon mixing, a timer was started, and the amount of recovered enzyme activity monitored at 2, 4, 6, 8, 10, 26, and 45 min intervals.

Figure 4

Thiol-mediated reactivation of oxidatively-inactivated SHP-2. (A) Oxidized SHP-2 (11 nM) was incubated with various thiols in Buffer A containing the chromogenic PTP substrate pNPP (20 mM) pNPP, pH 7 at room temperature. The time courses monitored the increase in absorbance at 410 nm resulting from the SHP-2-catalyzed release of p-nitrophenol from the substrate. Monothiols were 50 mM and dithiols 25 mM: DTT (), TGA OMe (), TGA (X), 2 ME (), GSH (*), NAC (+), no thiol (). (B) Instantaneous rates of reactivation (slopes) versus time. Data were fit with pseudo-first order kinetic treatment to give the following estimates of the apparent bimolecular rate constants calculated in units of M⁻¹ s⁻¹: DTT = 0.89, TGA-OMe = 0.44, TGA = 0.10, 2-ME = 0.02, GSH = 0.02, NAC = 0.01.

Figure 5

Reactivation of oxidatively inactivated SHP-2 by DTT and GSH. Assays were conducted as described in the Experimental Section. A. DTT-mediated recovery of activity from oxidized PTP1B. B. Pseudo first order rate constants (s⁻¹ × 10³) were plotted against DTT concentration (mM), affording a straight line of slope k_obs (M⁻¹ s⁻¹) which passes through the origin. This data is consistent with a bimolecular process in the rate determining step. C. GSH-mediated recovery of activity from oxidized SHP-2. D. Plotting the observed pseud first-order rate constants versus concentration of GSH affords a curvilinear (parabolic) graphical form, indicating a non-second order kinetic process. E. Plotting of the observed pseudo first-order rate constants versus the square of the GSH concentration affords a linear form, which passes through the origin. This suggests a process second-order in thiol concentration in the rate-determining step, with the relevant rate constant being equal to 0.29 ± 0.02 M⁻² s⁻¹.

Scheme 1

Oxidative inactivation of protein tyrosine phosphatases.

Scheme 2

Thiol-mediated recovery of catalytic activity from oxidized protein tyrosine phosphatases.

Scheme 3

Scheme 4

Scheme 4

Scheme 5

Proposed disulfide relay initiated by oxidative inactivation of SHP-2 (Panel A) and proposed mechanism for thiol-dependent recovery of activity from oxidized SHP-2 (Panel B).