Redox regulation of protein tyrosine phosphatase 1B by peroxymonophosphate (=O3POOH).

Reversible phosphorylation of tyrosine residues serves as a biochemical “switch” that alters the functional properties of many proteins involved in cellular signal transduction processes.1,2 The phosphorylation status of tyrosine residues in target proteins is controlled by the opposing actions of protein tyrosine kinases that catalyze the addition of phosphoryl groups and protein tyrosine phosphatases (PTPs) that catalyze their removal.2 Thus, PTPs play a central role in the regulation of diverse cellular processes including glucose metabolism, cell cycle control, and immune responses.3 The catalytic activities of many tyrosine kinases and phosphatases are strictly regulated to control the intensity and duration of cellular responses to various stimuli.2,4 For example, exposure of cells to insulin, growth factors, or cytokines activates kinases that add phosphoryl groups to tyrosine residues on target proteins.4 In some cases, this kinase action is potentiated by a rapid (2-5 min onset), transient inactivation of the tyrosine phosphatases that are responsible for removal of these phosphoryl groups.5,6 This involves downstream activation of NADPH oxidases that produce an intracellular burst of H2O2. The H2O2, in turn, leads to inactivation of select PTPs via oxidation of their catalytic cysteine thiol residues to the sulfenic acid oxidation state (Scheme 1).5,7 Oxidative inactivation of PTPs inside cells is transient because thiol-mediated reduction of the oxidized cysteine residue slowly regenerates the active form of the enzyme.5-7 Interestingly, despite clear evidence for its involvement in the intracellular regulation of PTPs, in vitro experiments reveal that H2O2, at physiological concentrations, is a rather sluggish PTP inactivator.5 Specifically, based upon the reported5 rate constants for in vitro inactivation of PTPs by H2O2 (e.g., k ) 9 M-1 s-1 for PTP1B), one can calculate that the half-life for inactivation of these enzymes by a steady-state concentration of 1 μM H2O2 will be approximately 20 h.8,9 For some applications, it may be desirable to identify small molecules that mimic the ability of hydrogen peroxide to effect transient, thiol-reversible, oxidative inactivation of PTPs. In general, PTP inactivators have potential both as therapeutic agents and as tools for the study of signal transduction pathways.10 Here, we set out to develop a redox regulator of PTP activity that is more potent than hydrogen peroxide. Toward this end, we envisioned that peroxymonophosphate (1, Scheme 1) might be an exceptional PTP inactivator, in which noncovalent association of the phosphoryl group with the highly conserved phosphate binding pocket found at the active site of all PTPs3 would serve to guide the peroxyl moiety into position for efficient reaction with the catalytic cysteine residue (inset, Scheme 1). In the studies described here, we employed the catalytic subunit of human PTP1B (aa 1-322) as an archetypal member of the PTP family of enzymes.3 Peroxymonophosphate (1) was prepared via electrolysis of potassium phosphate to generate peroxydiphosphate, followed by acid hydrolysis.11 The resulting peroxymonophosphate was characterized by 31P NMR and mass spectrometry. We find that peroxymonophosphate is, indeed, a superior inactivator of PTP1B with a KI of 6.6 ( 1.5 × 10-7 M and a kinact of 0.043 ( 0.008 s-1 (Figure 1A). Thus, peroxymonophosphate (kinact/KI ) 65 553 M-1 s-1) is over 7000 times more potent than H2O2 (9 M-1 s-1) as a PTP1B inactivator. The saturation kinetics observed for the inactivation of PTP1B by peroxymonophosphate offers evidence that the phosphoryl group of peroxymonophosphate does, indeed, provide noncovalent binding affinity for the enzyme active site. In contrast, H2O2 does not possess noncovalent affinity for PTP1B and inactivates the enzyme via a simple second-order reaction process.5 Consistent with a process involving covalent modification of the enzyme, inactivation of PTP1B by peroxymonophosphate is timedependent and is not reversed by gel filtration of the inactivated enzyme. The presence of the competitive PTP1B inhibitor phosphate13 markedly slows inactivation by peroxymonophosphate, indicating that the process is active site directed. Significantly, inactivation of PTP1B (1 μM, 1 min) by peroxyphosphate is readily reversed (88% recovery of activity) by treatment of the inactivated enzyme with dithiothreitol (Figure 1B). Together, the results suggest that peroxymonophosphate inactivates PTP1B via conversion of the active site cysteine residue to the sulfenic acid oxidation state, as shown Scheme 1.14 In principle, PTP1B could catalyze the hydrolysis of peroxymonophosphate to H2O2 and inorganic phosphate. However, two lines of evidence suggest that such an enzyme-catalyzed decomposition of peroxymonophosphate does not occur. First, addition of the H2O2-destroying enzyme catalase has no effect on the inactivation of PTP1B by peroxymonophosphate. Second, titration of the enzyme with peroxymonophosphate reveals a turnover number15 of approximately one (Figure 2), indicating that a single § Department of Chemistry. ‡ Department of Biochemistry. ⊥ Department of Veterinary Pathobiology and Veterinary Medical Diagnostic Laboratory. Scheme 1 Published on Web 04/06/2007