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, 20 (2), 444-7

Protection of a Single-Cysteine Redox Switch From Oxidative Destruction: On the Functional Role of Sulfenyl Amide Formation in the Redox-Regulated Enzyme PTP1B

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Protection of a Single-Cysteine Redox Switch From Oxidative Destruction: On the Functional Role of Sulfenyl Amide Formation in the Redox-Regulated Enzyme PTP1B

Santhosh Sivaramakrishnan et al. Bioorg Med Chem Lett.

Abstract

Model reactions offer a chemical mechanism by which formation of a sulfenyl amide residue at the active site of the redox-regulated protein tyrosine phosphatase PTP1B protects the cysteine redox switch in this enzyme against irreversible oxidative destruction. The results suggest that 'overoxidation' of the sulfenyl amide redox switch to the sulfinyl amide in proteins is a chemically reversible event, because the sulfinyl amide can be easily returned to the native cysteine thiol residue via reactions with cellular thiols.

Figures

Figure 1
Figure 1
Reaction of 2 with 10 equiv 2-mercaptoethanol in aqueous buffer: A) Compound 2 alone in buffer B) 1 min after addition of thiol C) 25 min after addition of thiol. Compound 2 (2.5 µL of a 10 mM stock in CH3CN) was added to a mixture containing sodium phosphate buffer (50 µL, 500 mM, pH 7.0), water (275 µL), 2-mercaptoethanol (25 µL of a 10 mM stock in water), and acetonitrile (147.5 µL) at 25 °C (final concentrations: 2, 50 µM; buffer, 50 mM, pH 7.0; thiol, 500 µM; acetonitrile, 30% by volume). The disappearance of 2 was monitored at 254 nm. Aliquots (40 µL) from the reaction mixture were injected onto a C-18 Varian Microsorb-MV column, 100 Å sphere size, 5 µm pore size, 25 cm length, 4.6 mm i.d. eluted with a solvent system composed of water with 0.5% acetic acid v/v (A) and acetonitrile (B), at a flow rate of 0.8 mL/min. The column was eluted with 70:30 A/B for 4 min and then ramped to 50:50 A/B over 4 min, held at 50:50 A/B for 7 min, and then ramped back to 70:30 A/B over the next 3 min. The peak at 10.2 min was identified as the mixed disulfide 4. The identity of 4 was confirmed by co-injection with an authentic standard and by LC/MS analysis which showed that the compound displays an m/z of 316 corresponding to that expected for the [M+H]+ ion of 4. The early peaks in the chromatogram correspond to buffer salts, 2-mercaptoethanol, and the disulfide of 2-mercaptoethanol.
Figure 2
Figure 2
A representative plot for the disappearance of 2 in the presence of thiol. Compound 2 (2.5 µL of a 10 mM stock in CH3CN) was added to a mixture containing sodium phosphate buffer (50 µL, 500 mM, pH 7.0), water (150 µL), 2-mercaptoethanol (50 µL of a 10 mM stock) and acetonitrile (247.5 µL) at 25 °C. The mixture (final concentrations: 2, 50 µM; buffer, 50 mM, pH 7.0; thiol, 1 mM; acetonitrile, 50% by volume) was vortex mixed and the disappearance of 2 (a is the peak area at time = t and a0 is the peak area at time = 0) monitored by reverse phase HPLC at regular time intervals as described in the legend for Figure 1. From the slope of the plot, a pseudo-first-order rate constant of 5.5 × 10−3 s−1 (t1/2 = 2 min) at 1 mM thiol was obtained. This corresponds to an apparent second-order rate constant of 5.5 ± 0.2 M−1 s−1.
Figure 3
Figure 3
Representative plot for the disappearance of 2 in the absence of thiol. Compound 2 (2.5 µL of a 10 mM stock in CH3CN) was incubated at 25 °C in a solution composed of sodium phosphate buffer (50 µL, 500 mM, pH 7.0), water (200 µL) and acetonitrile (247.5 µL). The mixture (final concentrations: 2, 50 µM; buffer, 50 mM, pH 7.0; acetonitrile, 50% by volume) was vortex mixed and the disappearance of compound 2 (a is the peak area at time = t and a0 is the peak area at time = 0) analyzed by reverse phase HPLC as described in the legend of Figure 1. The pseudo-first-order rate constant of 0.027 ± 0.001 min−1 was obtained from the slope of the plot. This corresponds to a half-life of 26 min for the hydrolysis of 2 under these conditions.
Scheme 1
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