Oxidative inactivation of protein tyrosine phosphatase 1B by organic hydroperoxides

Article abstract of DOI:10.1016/j.bmcl.2008.06.029

Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that play a central role in cell signaling. Organic hydroperoxides cause thiol-reversible, oxidative inactivation of PTP1B in a manner that mirrors the endogenous signaling agent hydrogen

Full text of DOI:10.1016/j.bmcl.2008.06.029

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5856–5859
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Oxidative inactivation of protein tyrosine phosphatase 1B by organic
hydroperoxides
Sanjib Bhattacharya, Jason N. LaButti, Derrick R. Seiner, Kent S. Gates *
Departments of Chemistry and Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that play a central role in cell sig-
naling. Organic hydroperoxides cause thiol-reversible, oxidative inactivation of PTP1B in a manner that
mirrors the endogenous signaling agent hydrogen peroxide.
Received 15 April 2008
Revised 10 June 2008
Accepted 10 June 2008
Available online 13 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Protein tyrosine phosphatase
Peroxides
Hydroperoxides
Enzyme inactivation
Protein tyrosine phosphatases (PTPs) are cysteine-dependent
enzymes that catalyze the hydrolytic removal of phosphate groups
from tyrosine residues in proteins (Scheme 1).1 Thus, PTPs, in
concert with protein tyrosine kinases, play a central role in cell sig-
naling by regulating the phosphorylation status and, in turn, the
functional properties of target proteins in various signal transduc-
rates of inactivation versus concentration, indicating that the inac-
tivation reaction is a second-order process (Fig. 1B). The slope of
–3
4
À1 À1
the line reveals a rate constant of 2.1 ± 0.2 Â 10 M
s
for the
reaction of 1 with PTP1B. For comparison, hydrogen peroxide inac-
À1 À1 10
tivates PTP1B with a rate constant of 10 M
s
.
The inactivation of PTP1B (250 nM) by 1 (1
lM, 3 min) is not re-
4
,5
tion pathways.
The cellular activity of some PTPs is regulated by hydrogen per-
oxide (H ) that is produced as a second messenger in response to
extracellular stimuli such as insulin, epidermal growth factor, and
versed upon removal of excess inactivator by gel filtration of the
enzyme through G25 Sephadex. The inactivation process is signif-
icantly slowed in the presence of phosphate ion, which is an active
2 2
O
site-directed inhibitor of the enzyme (K
i
= 17 mM). Specifically,
platelet-derived growth factor.6
tion of the active site cysteine thiol residue to a sulfenic acid.
some cases, the cysteine sulfenic acid undergoes subsequent con-
–9
H
2
O
2
inactivates PTPs via oxida-
treatment of the enzyme with 1 (15 M) for 15 s inactivates 88%
l
6
,10
In
of the enzyme under standard conditions, while in the presence
of phosphate ion (50 mM) only 49% inactivation is observed. To-
gether, the results suggest that 1 inactivates PTP1B via covalent
modification of the active site.
11–13
version to an active site sulfenyl amide linkage
or disul-
1
4,15
fide.
This type of oxidative inactivation of PTPs is slowly
reversed upon reaction of the inactivated enzyme with biological
Inactivation of PTP1B by 1 can be reversed by treatment of the
enzyme with thiols (Fig. 2). Specifically, inactivation of PTP1B
thiols (Scheme 1).1
0–15
Given the biological importance of PTPs, it may be useful to
identify organic reagents that regulate PTP activity through redox
1
6–20
mechanisms.
With this in mind, we examined the ability of
O
O
several organic hydroperoxides (1–4) to effect thiol-reversible, oxi-
dative inactivation of the archetypal member of the PTP family,
PTP1B. We find that peracetic acid (1) is a potent, time-depen-
dent inactivator of the catalytic subunit of PTP1B (aa 1–322)
Fig. 1A). The observation of time-dependent inactivation is consis-
tent with a process involving covalent modification of the en-
zyme. A good linear fit is obtained in a plot of the apparent
O
OH
Cl
OH
O
OH
O
2
1
O
CO2—
1
2
3
6
(
2
2
O
OH
OOH
O
OH
O
*
Corresponding author. Tel.: +1 573 882 6763; fax: +1 573 882 2754.
E-mail address: gatesk@missouri.edu (K.S. Gates).
4
5
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.029

S. Bhattacharya et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5856–5859
5857
Substrate
O
Substrate
O
H
O
—O
HO
HN
O
OH
H
O—
+
NH
O—
P
H N
2
O
O
S—
O—
P
O
O
O—
NH
NH
O
S
H
N
H
N
H
N
H
N
HN
HN
O
O
O
O
O
RSSR
H O
2
2
2
RSH
O
O
—O —P OH
O
HO
O
OH
NH
O
S
O
H
N
NH
O
S—
H
H
N
HN
O
N
H
N
O
HN
O
O
inactive enzyme
Scheme 1.
1
9,20
Figure 1. Rate of inactivation of PTP1B by compound 1. (A) Progress curves for the inactivation of PTP1B by 1. PTP1B was isolated as described previously
and thiol-free
samples of the enzyme were prepared by gel filtration of the protein through G25 Sephadex immediately prior to use according to reported procedures.1
9,20
Thiol-free PTP1B
(
25 nM final) was added to a solution of 1 (250 nM–2 lM) in 3,3-dimethyl glutarate buffer (50 mM, pH 7.0) containing the substrate p-nitrophenyl phosphate (p-NPP, 20 mM)
23
at 23 °C. The apparent rate constant (kobs) for each concentration was calculated by the method of Voet and co-workers. (B) Determination of the second-order rate constant
for the inactivation of PTP1B by 1. The apparent (pseudo-first-order) rate constants for inactivation of PTP1B by 1 were plotted versus concentration and the second-order rate
constant for inactivation of PTP1B by 1 was extracted from the slope. Inactivator concentrations >10-times the enzyme concentration were used in the plot. The concentration
of 1 (and other peroxides) in stock solutions was determined by titration.24
(
750 nM) with low concentrations of 1 (800 nM, 10 min, 23 °C), fol-
lowed by treatment of the inactive enzyme with dithiothreitol
DTT, 100 mM final concentration), led to substantial return of
Two possible mechanisms can be considered for the inactiva-
tion of PTP1B by 1. Direct reaction of the active site cysteine resi-
due with 1 would yield the inactive, sulfenic acid form of the
(
2
6–28
enzymatic activity (73% of initial activity). (When enzyme is not
treated by 1, essentially all activity is retained under these condi-
tions). This suggests that reaction of PTP1B with 1 predominantly
converted the active site cysteine residue to the sulfenic acid oxi-
dation state. In contrast, following treatment of the enzyme with
enzyme (Scheme 2A).
ton-type reaction could generate diffusible oxygen radicals that
Alternatively, a metal-mediated Fen-
2
9
3
0
might oxidize the active site cysteine residue. Two lines of exper-
imentation argue against a Fenton-type process in the inactivation
of PTP1B by 1. First, we find that addition of the classical oxygen
radical scavenging agent mannitol has little effect on the inacti-
vation of PTP1B by 1. Second, addition of FeSO (5 M) inhibits,
2
9
higher concentrations of 1 (5
original activity returned upon treatment with DTT. Under these
lM, 10 min, 23 °C), only 10% of the
4
l
conditions, the active site cysteine presumably underwent signifi-
rather than facilitates, the inactivation process, presumably due
to the fact that Fe(II) mediates destruction of the peracid.29 With
the Fenton-type mechanism excluded, it is reasonable to sus-
2
cant amounts of ‘over-oxidation’ to the sulfinic (RSO H) or sulfonic
acid (RSO H) oxidation states that are not readily reduced to active
3
2
6–28
enzyme by DTT. It is known that cysteine residues in redox
regulated enzymes can be irreversibly ‘over-oxidized’ to sulfinic
and sulfonic acids by hydrogen peroxide under some
pect
that the inactivation of PTP1B by 1 proceeds via the
mechanism shown in Scheme 2A.
We find that aromatic peracids 2 and 3 inactivate PTP1B even
more effectively than 1 (Fig. 3). For example, 2 and 3, at 150 nM
1
2,14,25
conditions.

5
858
S. Bhattacharya et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5856–5859
À1 À1
4
inactivates PTP1B with a rate constant of 20.3 ± 0.8 M
s
. This
À1 À1
is comparable to the literature rate of 10 M
s
reported for
1
0
hydrogen peroxide. Analogous to the inactivation of PTP1B by 1
and hydrogen peroxide, inactivation by 4 (50 M, 10 min) is re-
l
versed upon reaction of the enzyme with DTT (54% return of activ-
ity, following treatment with 100 mM DTT for 1 h).
In summary, we find that peracetic acid (1) is a potent oxidative
inactivator of PTP1B. At low concentrations of 1 (61 lM), enzyme
activity was readily recovered by treatment of the inactivated en-
zyme with thiol. Overall, the results suggest that 1 inactivates the
enzyme via oxidation of the active site cysteine thiol residue to the
sulfenic acid oxidation state. Peracetic acid (1) is approximately
2000 times more potent than hydrogen peroxide, a known endog-
enous regulator of cellular PTP activity. The superior activity of 1 is
likely due to the intrinsically higher reactivity of peracids com-
Figure 2. Inactivation of PTP1B by 1 can be reversed by addition of dithiothreitol
DTT). Thiol-free PTP1B (25 nM) was added to a solution of 1 (1 M) in 3,3-dimethyl
glutarate buffer (50 mM, pH 7.0) containing the substrate p-nitrophenyl phosphate
(
l
(p-NPP, 20 mM) at 23 °C. When inactivation of the enzyme was nearly complete
2
8
pared to hydrogen peroxide. Similarly, the superior inactivating
(
300 s), a concentrated stock solution of DTT in water was added to give a final
concentration of 20 mM. The experiment was conducted in a quartz cuvette and
enzyme activity was measured by monitoring the enzyme-catalyzed release of p-
nitrophenolate ion from the substrate p-NPP at 410 nm.
power of 2 and 3 compared to 1 likely reflects their higher intrinsic
3
3
reactivity as oxidants. In short, the trends reported here provide
the useful predictive observation that the PTP-inactivating power
of sterically unhindered organic peroxides will track closely with
À
the leaving group ability of the substituent RO that is displaced
by thiol attack on the peroxide (Scheme 2A). In addition, inactiva-
tion by peracids such as 2 and 3 may be fast because the aromatic
rings in these compounds bind noncovalently to PTP1B prior to the
inactivation reaction via interactions with phosphotyrosine-bind-
ing residues such as Tyr46 and Phe182.
S—
HO OR
Fe(II)
S OH +
—OR
A
B
+
HO OR
+
HO— + •OR + Fe(III)
Tanner and Denu previously reported that tert-butyl hydroper-
Scheme 2. Potential mechanisms for PTP inactivation by hydroperoxides.
10
oxide (5) and cumene hydroperoxide (6) do not inactivate PTPs.
As part of this work, we confirmed their findings (data not shown).
These hydroperoxides react with small-molecule thiols at rates
concentrations, completely inactivate the enzyme within seconds.
In contrast, when the enzyme is treated with the same concentra-
tion of 1, a significant amount of enzyme activity remains at 1 min
3
4
comparable to hydrogen peroxide ; therefore, it seems likely that
their failure to inactivate PTPs stems, not from an inherent lack of
reactivity, but from the fact that the substituents on these tertiary
hydroperoxides form an ‘umbrella’ of steric bulk that prevents the
peroxyl residue from penetrating the deep active site cavity of
PTP1B to reach the catalytic cysteine residue. Indeed, as part of this
study, we showed that the less hindered hydroperoxide 4 inacti-
vates PTP1B with potency comparable to hydrogen peroxide. Evi-
dently, the planar peracids 1–3 clearly are not sterically
precluded from entering the enzyme’s active site.
(
Fig. 3). For another point of comparison, 150 nM H
surable effect on enzyme activity under these conditions. Indeed
concentrations of H approaching 100 M are required to yield
2 2
O has no mea-
2
O
2
l
inactivation rates comparable to 150 nM 1 (Fig. 3). Finally, it is
noteworthy that 1–3 can inactivate the enzyme in the presence
of the biological thiol, glutathione.31 For example, treatment of
PTP1B with 1 (5 lM, 15 s) causes a 29% loss of enzyme activity
even in the presence of 1 mM glutathione.
Our observation that organic peroxides have the ability to cause
thiol-reversible, oxidative inactivation of PTPs has some significant
implications. Inactivation of PTPs could contribute to the biological
activity of some hydroperoxide-containing natural products.35 Fur-
thermore, a variety of organic hydroperoxides including lipid and
In addition, we examined the ability of 2-hydro-
peroxytetrahydrofuran (4) to inactivate PTP1B. This reagent was
synthesized by the method of Gold and co-workers.3 We find that
2
3
6,37
amino acid hydroperoxides are generated in cells.
Indeed,
while our work was in progress, Davies and co-workers reported
the inactivation of PTP1B by amino acid hydroperoxides whose
3
8
chemical structures, as yet, remain undefined. Our work provides
quantitative measurement of the PTP-inactivating power of several
structurally well-defined organic hydroperoxides. In addition, it is
interesting to note that 1 and other peracids are produced by a
3
9–43
number of bacterial and eukaryotic enzymes.
This offers the
possibility that peracids could be involved in the physiological or
pathophysiological regulation of PTPs and other cysteine-depen-
dent enzymes. Overall, our work provides new evidence that
endogenous and exogenous organic hydroperoxides have the po-
tential to regulate or dysregulate cellular signaling pathways. Con-
tinued investigation of PTP inactivation by this class of molecules is
warranted.
Figure 3. Comparison of the inactivation of PTP1B by compounds 1, 2, 3, and
hydrogen peroxide. Thiol-free PTP1B (25 nM) was added to a cuvette containing
3
,3-dimethyl glutarate buffer (50 mM, pH 7.0), p-NPP (10 mM), and the hydroper-
Acknowledgments
oxide of interest (1, 2, 3, or H ) at 24 °C. Enzyme inactivation progress curves
2 2
O
showing the amount of PTP1B activity remaining as a function of time were
obtained by monitoring the enzyme-catalyzed release of p-nitrophenolate ion from
the substrate p-NPP at 410 nm. Note: curves for control ‘no inactivator’ and 150 nM
We thank the National Institutes of Health for financial support
of this work (CA 83925 and CA 119131) and the reviewers for help-
ful comments.
2 2
H O are overlapping.

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5859
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