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

Article abstract of DOI:10.1021/ja070194j

Reversible phosphorylation of tyrosine residues serves as a biochemical "switch" that alters the functional properties of many proteins involved in cellular signal transduction processes. Protein tyrosine phosphatases (PTPs) catalyze the removal of phosph

Full text of DOI:10.1021/ja070194j

Published on Web 04/06/2007
Redox Regulation of Protein Tyrosine Phosphatase 1B by
Peroxymonophosphate (dO3POOH)
Jason N. LaButti,§ Goutam Chowdhury,§ Thomas J. Reilly, and Kent S. Gates*,§,‡
Department of Chemistry, Department of Biochemistry, and Department of Veterinary Pathobiology and Veterinary
Medical Diagnostic Laboratory, UniVersity of Missouri, Columbia, Missouri 65211
Received January 10, 2007; E-mail: gatesk@missouri.edu
Scheme 1
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 respon-
sible for removal of these phosphoryl groups.5,6 This involves
downstream activation of NADPH oxidases that produce an
intracellular burst of H2O2.7 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).
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 time-
dependent and is not reversed by gel filtration of the inactivated
enzyme. The presence of the competitive PTP1B inhibitor phos-
phate13 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 peroxy-
monophosphate to H2O2 and inorganic phosphate. However, two
lines of evidence suggest that such an enzyme-catalyzed decom-
position 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
In the studies described here, we employed the catalytic subunit
of human PTP1B (aa 1-322) as an archetypal member of the PTP
§ Department of Chemistry.
‡ Department of Biochemistry.
Department of Veterinary Pathobiology and Veterinary Medical Diagnostic
Laboratory.
9
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J. AM. CHEM. SOC. 2007, 129, 5320-5321
10.1021/ja070194j CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
intracellular signaling agent, H2O2 is produced inside cells as a
byproduct of aerobic metabolism and as a result of various disease
states.7,20 Phosphoryl transfer to oxygen nucleophiles is a ubiquitous
reaction in biology21 and H2O2, though present at relatively low
concentrations, is a substantially better nucleophile than water.22 It
remains to be seen whether peroxymonophosphate can be generated
through spontaneous or enzyme-catalyzed reactions of H2O2 with
phosphoryl donors under physiological conditions; however, the
results reported here suggest that nanomolar concentrations of
peroxymonophosphate could effect reversible down-regulation of
cellular PTP activity within minutes. In this regard, peroxymono-
phosphate possesses key properties expected for an endogenous
signaling molecule involved in the redox regulation of PTP activity.
Figure 1. (A) Inactivation progress curves showing time-dependent
inactivation of PTP1B by peroxymonophosphate (1). PTP1B (25 pmol) was
incubated with various concentrations of 1 (250-1500 nM) at 25 °C in
aqueous buffer (50 mM Tris, 50 mM bis-Tris, 100 mM NaOAc, pH 7)
containing the substrate p-nitrophenylphosphate (p-NPP, 10 mM), and
enzyme-catalyzed release of p-nitrophenolate ion from the substrate was
monitored at 410 nm. The inactivation constants, kinact, and KI were
calculated from the data as described by Voet and co-workers12 (see
Supporting Information. (B) Reaction progress curves showing inactivation
of PTP1B by 1 and reactivation of the inactive enzyme by treatment with
thiol. The enzyme PTP1B (100 pmol) was inactivated by 1 (2 µM) in
aqueous buffer (50 mM Tris, 50 mM bis-Tris, 100 mM NaOAc, pH 7) at
25 °C containing the substrate p-NPP (10 mM). When the enzyme
inactivation was complete, dithiothreitol (DTT) was added to a final
concentration of 5 mM. Control experiments show that DTT does not react
with p-NPP.
Acknowledgment. We are grateful to the National Institutes
of Health for partial support of this work (CA 83925, CA 100757,
and CA 119131). We thank Professors Nicholas Tonks and Jonathan
Chernoff for providing PTP1B expression vectors.
Supporting Information Available: Experimental protocols for
all experiments. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Figure 2. Turnover number for the inactivation of PTP1B by peroxy-
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Mammalian cells contain millimolar concentrations of thiols that
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inactivation of PTP1B (21 ( 3% activity remaining) within 5 min
even in the presence of physiologically relevant concentrations of
glutathione (1 mM). This result shows that peroxymonophosphate
reacts selectively with the active site cysteine thiolate of PTP1B
over solution thiols.
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oxidative inactivator of the protein tyrosine phosphatase PTP1B.
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physiologically relevant concentrations of the biological thiol
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