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Published: September 13, 2011
r 2011 American Chemical Society 15803 dx.doi.org/10.1021/ja2077137 | J. Am. Chem. Soc. 2011, 133, 15803–15805
COMMUNICATION
pubs.acs.org/JACS
The Biological Buffer Bicarbonate/CO2 Potentiates H2O2-MediatedInactivation of Protein Tyrosine PhosphatasesHaiying Zhou,§ Harkewal Singh,§ Zachary D. Parsons,§ Sarah M. Lewis,§ Sanjib Bhattacharya,§
Derrick R. Seiner,§ Jason N. LaButti,§ Thomas J. Reilly,† John J. Tanner,*,§,‡ and Kent S. Gates*,§,‡
§Department of Chemistry, ‡Department of Biochemistry, and †Department of Veternary Pathobiology andVeterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, Missouri 65211, United States
bS Supporting Information
ABSTRACT: Hydrogen peroxide is a cell signaling agentthat inactivates protein tyrosine phosphatases (PTPs) viaoxidation of their catalytic cysteine residue. PTPs areinactivated rapidly during H2O2-mediated cellular signaltransduction processes, but, paradoxically, hydrogen per-oxide is a rather sluggish PTP inactivator in vitro. Here wepresent evidence that the biological buffer bicarbonate/CO2
potentiates the ability of H2O2 to inactivate PTPs. Theresults of biochemical experiments and high-resolutioncrystallographic analysis are consistent with a mechanisminvolving oxidation of the catalytic cysteine residue byperoxymonocarbonate generated via the reaction of H2O2
with HCO3�/CO2.
Hydrogen peroxide is a signaling agent that mediates cellularresponses to growth factors, hormones, and cytokines such
as platelet-derived growth factor, epidermal growth factor, vas-cular endothelial growth factor, insulin, tumor necrosis factor-α,and interleukin-1β.1,2 Protein tyrosine phosphatases (PTPs) areimportant targets of H2O2 produced during signal transductionprocesses.2,3 PTPs help regulate a variety of critical mammaliansignaling pathways by catalyzing the removal of phosphorylgroups from phosphotyrosine residues on target proteins. H2O2
inactivates PTPs via oxidation of the catalytic cysteine residue inthese enzymes, leading to elevated levels of tyrosine phosphory-lation on key signaling proteins.3�7 Reactions with cellularthiols ultimately return oxidized PTPs to the catalytically activeforms.3�6,8
Peroxide-mediated inactivation of intracellular PTPs duringsignaling events typically occurs rapidly (5�15 min).5,9,10 Para-doxically, the rate constants measured for in vitro inactivationof purified PTPs by H2O2 (e.g., 10�40 M�1 s�1 for PTP1B)4
suggest that the loss of enzyme activity should be rather sluggish(t1/2 = 5�200 h) at the low cellular concentrations of H2O2
thought to exist during signaling events (0.1�1 μM).11,12 Thiskinetic discrepancy led us and others to consider the possibilitythat H2O2may undergo spontaneous or enzymatic conversion tomore reactive oxidizing agents that effect rapid intracellular in-activation of PTPs.11�14
Along these lines, we set out to explore a potential role for thebiological bicarbonate/CO2 buffer system in H2O2-mediatedsignal transduction. H2O2 reacts with bicarbonate/CO2 to generatethe highly reactive oxidant, peroxymonocarbonate (Figure 1a).15�17
This process may be catalyzed by thiols and sulfides.15�17 Peroxy-monocarbonate is an acyl peroxide,18 and on the basis of our recentstudies of organic acyl peroxides,13 we anticipated that this speciesmight be a potent PTP inactivator.
To address this question, we employed the catalytic domain(aa 1�322) of recombinant human PTP1B, as an archetypalmember of the PTP family. First, we confirmed that H2O2 alonecauses time-dependent inactivation of PTP1B with an apparentsecond-order rate constant of 24 ( 3 M�1 s�1 at 25 �C, pH 7.4
The extracellular and intracellular concentrations of bicarbonateare 25 and 14.4 mM, respectively,16 and we examined the effectsof bicarbonate within this general concentration range. We foundthat the presence of potassium bicarbonate markedly increasedthe rate of time-dependent PTP1B inactivation by H2O2. Forexample, potassium bicarbonate increased the apparent second-order rate constants for inactivation of PTP1B byH2O2 to 202(4 and 330 ( 11 M�1 s�1 at concentrations of 25 and 50 mM,respectively (25 �C, pH 7, Figure 2). At physiological tempera-ture (37 �C), the rate of inactivation by the H2O2�KHCO3 sys-tem increases further to 396 ( 10 M�1 s�1 (KHCO3, 25 mM,pH 7, Figure S9). Other bicarbonate salts (NaHCO3 and NH4H-CO3) produced similar effects (Figure S21). Preincubation ofH2O2
and KHCO3 (up to 2 h) prior to addition of the enzyme did notsignificantly alter the observed rate of inactivation (Figure S10).Control experiments showed that KCl (25mM), NaCl (25mM),
Figure 1. (a) Possible mechanism for the inactivation of PTP1B byH2O2�HCO3
�. (b) Treatment of PTP1B with H2O2�HCO3� yields
the oxidized, sulfenyl amide form of the enzyme. The structure of theoxidized enzyme was solved at 1.7 Å resolution (pdb code 3SME). Theimage shows a simulated annealing σA-weighted Fo � Fc omit mapcontoured at 3.0σ, covering Cys215 and flanking residues.
Received: August 15, 2011
15804 dx.doi.org/10.1021/ja2077137 |J. Am. Chem. Soc. 2011, 133, 15803–15805
Journal of the American Chemical Society COMMUNICATION
or MgCl2 (2 mM) did not significantly alter the rate at whichH2O2 inactivated PTP1B (Figures S11�S13). This indicatesthat the effect of bicarbonate on the peroxide-mediated inactiva-tion of PTP1B was not merely an ionic strength effect. It is im-portant to emphasize that KHCO3 alone did not cause time-dependent inactivation of PTP1B at 24 �C (Figure 2a). Thetime-dependent nature of the inactivation observed here is con-sistent with a process involving covalent chemical modificationof the enzyme.
The cellular milieu contains millimolar concentrations of thiolssuch as glutathione, which can decompose peroxides.12 There-fore, we examined the effects of glutathione on the inactivat-ion of PTP1B by H2O2�KHCO3. We find that the H2O2�KHCO3 system causes rapid and complete loss of enzymeactivity in the presence of glutathione (1 mM), with only anapproximately 2-fold decrease in the observed rate of inactiva-tion (Figure 3a).
Time-dependent inactivation of PTP1B by the H2O2�KHCO3
systemwas slowed by competitive inhibitors (Figures S14 and S15).For example, phosphate (50 mM) slowed inactivation by a factorof 1.7 ( 0.1. Activity did not return to the inactivated enzymefollowing gel filtration or dialysis to remove H2O2 and KHCO3
(Figures S22 and S23). These results suggest that inactivationof PTP1B by H2O2�KHCO3 involves covalent modification ofan active-site residue. Catalytic activity was recovered upon treat-ment of the inactivated enzyme with thiols such as dithiothreitol(DTT, Figures 3b and S24). For example, when the enzyme wasinactivated by treatment withH2O2�KHCO3 (34 μMand 25mM,
respectively, for 3 min to yield 80% inactivation), almost all(98%) of the initial activity was recovered by treatment withDTT (50 mM, 30 min, 25 �C). The thiol-reversible nature of theinactivation reaction is consistent with a mechanism involvingoxidation of the enzyme’s catalytic cysteine residue.3,6,8 Indeed,crystallographic analysis of PTP1B treated with H2O2�KHCO3
produced a 1.7 Å resolution structure showing that the catalyticcysteine residue was oxidized to the cyclic sulfenyl amide residueobserved previously for this enzyme (Figure 1b and SupportingInformation).3,6�8 There was no evidence in the electron densitymap for oxidation at any other residue in the enzyme. Peroxy-carbonate can decompose to yield highly reactive oxygen radicalsin the presence of transition metals.16,19 However, addition ofradical scavengers or chelators of adventitious trace metals hadno effect on the inactivation of PTP1B by the H2O2�KHCO3
system (Figures S16�S18), suggesting that the enzyme inactiva-tion process described here proceeds via a two-electron oxidationmechanism as shown in Figure 1a.
We also examined the ability of bicarbonate to potentiateH2O2-mediated inactivation of SHP-2, a different member ofthe PTP enzyme family. The intracellular activity of SHP-2 isthought to be redox regulated in response to platelet-derivedgrowth factor, endothelin-1, and T-cell receptor stimulation.1
Our experiments employed the catalytic domain (aa 246�527)of the recombinant human enzyme. We found that the inactiva-tion of SHP-2 by H2O2 occurs with an apparent second-orderrate constant of 15 ( 2 M�1 s�1 (25 �C, pH 7, Figure S19).20
The presence of potassium bicarbonate (25 mM) increased theapparent second-order rate constant for inactivation of SHP-2to 167 ( 12 M�1 s�1, an 11-fold rate increase (Figure S20).Similar to PTP1B, the inactivation process was slowed by thecompetitive inhibitor sodium phosphate, and enzyme activitywas recovered by treatment of the inactivated enzyme with DTT(Figures S25 and S26).
Figure 2. Kinetics of PTP1B inactivation by H2O2�KHCO3. (a) Asemi-log plot showing time-dependent loss of PTP1B activity in thepresence of various concentrations of H2O2 (the lines correspond to 0,12, 23, 33, 45, 57, and 68 μM H2O2 from top to bottom) at a singleconcentration of KHCO3 (25 mM). Inactivation assays were carried outas described in the Supporting Information. The pseudo-first-order rateconstant for inactivation at each concentration of H2O2 (kobs) was cal-culated from the slope of the line. (b) The apparent second-order rateconstant for inactivation by the H2O2�KHCO3 (25 mM) system wasobtained from the slope of the replot of kobs values obtained from thedata shown in panel a versus H2O2 concentration. (c) Plots of kobs ver-sus H2O2 concentration in the presence of various concentrations ofKHCO3 (see also Figures S1�S8). This plot provides a graphical depic-tion of how, as KHCO3 concentration increases, markedly lower conc-entrations of H2O2 are required to achieve a given rate of PTP1B in-activation. (d) Plot of the apparent second-order rate constants for theinactivation of PTP1B by H2O2 in the presence of various concentra-tions of KHCO3.
Figure 3. Effect of added thiol on the inactivation of PTP1B by H2O2�KHCO3 and reactivation of inactivated enzyme by dithiothreitol. (a)The inactivation of PTP1B by H2O2�KHCO3 proceeds in the presenceof glutathione. Thiol-free PTP1B (16.7 nM) was inactivated by treat-ment with H2O2 (100 μM) and KHCO3 (25 mM) in buffer containingsodium acetate (100 mM), bis-Tris (50 mM, pH 7.0), Tris (50 mM),DTPA (50 μM), Tween-80 (0.0005%), and p-NPP (10 mM) in a quartzcuvette. Enzyme activity in the sample was continuously monitored byfollowing the release of p-nitrophenolate at 410 nm. Reactions containedthe indicated combinations of H2O2 (150 μM), glutathione (GSH,1 mM), and KHCO3 (25 mM). (b) Catalytic activity can be recoveredby treatment of inactivated enzyme with dithiothreitol (DTT). Enzymeactivity was continuously monitored as described above. Thiol-freePTP1B (16.7 nM) was inactivated by treatment with H2O2 (100 μM)and KHCO3 (25 mM) as described above. When enzyme inactivationwas complete, DTT (5 μL of a 1 M solution in H2O) was added directlyto the cuvette to yield a final concentration of 5 mM DTT, and therecovery of enzyme activity was measured by observing the turnover ofsubstrate at 410 nm (control reactions showed that DTT alone does notyield significant release of p-nitrophenolate).
15805 dx.doi.org/10.1021/ja2077137 |J. Am. Chem. Soc. 2011, 133, 15803–15805
Journal of the American Chemical Society COMMUNICATION
Finally, for the purposes of comparison, we measured theability of H2O2 alone, or the H2O2�KHCO3 system, to inacti-vate a different type of cysteine-dependent enzyme, papain. Wefound that H2O2 alone inactivates this cysteine protease with anobserved second-order rate constant of 43( 7M�1 s�1, whereasinactivation of papain by the H2O2�KHCO3 (25 mM) systemoccurs with an observed rate constant of 83 ( 9 M�1 s�1
(Figures S27 and S28). The∼2-fold enhancement in the rate ofpapain inactivation engendered by bicarbonate is modest com-pared to its effect on the oxidation of PTPs and is similar to the2-fold enhancement exerted by bicarbonate (25 mM) on theobserved rate of peroxide-mediated oxidation of low-molecular-weight thiols.21 The larger effect of bicarbonate on the H2O2-mediated inactivation of PTPs suggests that this may be a mech-anism-based inactivation process, in which the anion-bindingpocket and general acid�base residues at the active site of theenzyme22 catalyze oxidation of the active-site cysteine byH2O2�KHCO3.
In summary, we report that the biological buffer systembicarbonate/CO2 selectively potentiates the ability of H2O2 toinactivate PTPs. Bicarbonate/CO2 enables steady-state concen-trations of H2O2 in the low micromolar range to inactivate PTPswithin the biologically relevant time frame of 10�15 min. Still,the rate constants reported here do not rise to the levels reportedfor other cellular H2O2 sensors such as the peroxiredoxins.
3,12,23,24
Therefore, spontaneous conversion of the second messengerH2O2 to peroxymonocarbonate may work in tandem withcolocalization,25 compartmentalization, or other means26 of genera-ting localized H2O2 concentration gradients to effect rapid,transient down-regulation of selected PTPs during signal trans-duction processes. The chemistry described here, in some regards,is reminiscent of the abilities of superoxide and CO2 to modulatethe properties of the cell signaling agent nitric oxide.27
’ASSOCIATED CONTENT
bS Supporting Information. Methods and results of inacti-vation andmechanism experiments. Thismaterial is available freeof charge via the Internet at http://pubs.acs.org.
’AUTHOR INFORMATION
Corresponding Authorgatesk@missouri.edu; tannerjj@missouri.edu
’ACKNOWLEDGMENT
We thank the Diabetes Action Research and Education Foun-dation and the NIH for partial support of this work (KSGCA83925 and 119131). H.S. was supported by a predoctoralfellowship from National Institutes of Health grant DK071510and a Chancellor’s Dissertation Completion Fellowship from theUniversity of Missouri�Columbia. We thank Dr. Jay Nix ofALS beamline 4.2.2 for assistance with data collection. Part ofthis work was performed at the Advanced Light Source. TheAdvanced Light Source is supported by the Director, Office ofScience, Office of Basic Energy Sciences, of the U.S. Depart-ment of Energy under Contract No. DE-AC02-05CH11231.This paper is dedicated to Prof. Richard B. Silverman on theoccasion of his 65th birthday.
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K. S. Antioxid. Redox Signal. 2011, 15, 77–97.(4) (a) Denu, J. M.; Tanner, K. G. Biochemistry 1998,
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(8) Sivaramakrishnan, S.; Cummings, A. H.; Gates, K. S. Bioorg. Med.Chem. Lett. 2010, 20, 444–447.
(9) (a) Mahedev, K.; Zilbering, A.; Zhu, L.; Goldstein, B. J. J. Biol.Chem. 2001, 276, 21938–21942. (b) Kwon, J.; Lee, S.-R.; Yang, K.-S.;Ahn, Y.; Kim, Y. J.; Stadtman, E. R.; Rhee, S. G. Proc. Natl. Acad. Sci.U.S.A. 2004, 101, 16419–16424.
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(15) (a) Regino, C. A. S.; Richardson, D. E. Inorg. Chim. Acta 2007,360, 3971–3977. (b) Bonini, M. G.; Fernandes, D. C.; Augusto, O.Biochemistry 2004, 43, 344–351.
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(18) Adam, A.; Mehta, M. Angew. Chem., Int. Ed. 1998, 37, 1387–1388.
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(20) Chen, C.-Y.; Willard, D.; Rudolph, J. Biochemistry 2009, 48,1399–1409.
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S1
Supporting Information
The Biological Buffer, Bicarbonate/CO2, Potentiates H2O2-Mediated Inactivation of Protein Tyrosine Phosphatases
Haiying Zhou§, Harkewal Singh,§ Zachary Parsons§, Sarah M. Lewis§, Sanjib
Bhattacharya§, Derrick R. Seiner§, Jason N. LaButti§, Thomas J. Reilly†, John J.
Tanner,§,‡ ,* and Kent S. Gates§,‡,*
§Department of Chemistry, ‡Department of Biochemistry, and †Department of Veterinary
Pathobiology and Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO 65211
Contents 1. Materials…………………………………………….………………………...………S2 2. Supplemental method 1. Enzyme inactivation assays………………………….….....S5 3. Inactivation of PTP1B by H2O2 and by H2O2-KHCO3 mixtures.........……………S7-10 4. Inactivation of PTP1B by H2O2-KHCO3 at 37 ˚C.........…..…………………………S11 5. Effects of preincubation on the inactivation of PTP1B by H2O2-KHCO3……….......S11 6. Effects of NaCl, KCl, and MgCl2 on inactivation of PTP1B by H2O2………....S12-S13 7. Effects of the competitive inhibitors on inact. of PTP1B by H2O2-KHCO3……..S13-14 8. Effects of radical scavengers on the inactivation process ………...…………......S14-16 9. Inactivation of SHP-2 by H2O2…………………………………………………..…..S17 10. Inactivation of SHP-2 by H2O2-KHCO3 ……………………………………….......S17 11. Supplemental method 2. Continuous assay for inactivation of PTP1B by H2O2-
KHCO3, -NaHCO3, and -NH4HCO3 ………………………………………….S17-18 12. Supplemental method 3. Activity of PTP1B inactivated by H2O2-KHCO3 does not
return upon gel filtration…………………………………………………...….S18-19 13. Supplemental method 4. The activity of PTP1B inactivated by H2O2-KHCO3 does
not return upon dialysis……………………………………………………….S19-20 14. Supplemental method 5. Treatment of PTP1B inactivated by H2O2-KHCO3 with
dithiothreitol (DTT) restores catalytic activity of the enzyme……………..…S20-21 15. Supplemental method 6. Treatment of SHP-2 inactivated by H2O2-KHCO3 with
dithiothreitol (DTT) restores catalytic activity of the enzyme…………..........S21-22 16. The competitive inhibitor sodium phosphate slows the inactivation of SHP-2 by
H2O2-KHCO3……………...……………………..…………………………....S22-23 17. Supplemental method 7. Inactivation of papain……...............................................S23 18. Inactivation of papain by H2O2 and H2O2-KHCO3…….………………………..….S24 19. Supplemental method 8. Crystallographic analysis of PTP1B treated with H2O2-
KHCO3……………………………………………………………………..…S24-27 20. Supplemental Literature Cited…………………………………………………...…S28 *Department of Chemistry, 125 Chemistry Building, University of Missouri-Columbia, Columbia, MO 65211. E-mail: gatesk@missouri.edu and tannerjj@missouri.edu, phone: (573) 882-6763; (573) 884-1280; fax: (573) 882-2754
S2
Materials. Reagents were purchased from the following suppliers: sodium acetate,
tris(hydroxymethyl)aminomethane (Tris), 2-[bis-(2-hydroxyethyl)-amino]-2-
hydroxymethyl-propane-1,3-diol (Bis-tris), 4-nitrophenyl phosphate disodium salt
hexahydrate (PNPP), 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), L-glutathione reduced
(GSH), D,L-dithiothreitol (DTT), ammonium bicarbonate, ethylenediaminetetraacetic
acid, disodium salt dehydrate (EDTA), 2-[4-(2-hydroxyethyl)piperazin-1-
yl]ethanesulfonic acid (HEPES), diethylenetriaminepentaacetic acid (DTPA), sodium
hydrogen phosphate, papain (cat# P-3125), and Nα-benzoyl-L-arginine 4-nitroanilide
hydrochloride (cat# B3279) were obtained from Sigma-Aldrich (St. Louis, MO). Sodium
chloride, sodium hydroxide, potassium bicarbonate, sodium phosphate, and sodium
bicarbonate were obtained from Fisher. Zeba mini and micro centrifugal buffer exchange
columns, and Tween-80 were purchased from Pierce Biotechnology (Rockford, IL).
Amicon Ultra centrifugal filter devices were purchased from Millipore (Milford, MA).
The enzyme consisting of amino acids 1-322 of human PTP1B was expressed and
purified as described previously1 and the concentration of active enzyme in stock
solutions was determined as described by Pregel et al.2 “Thiol-free” PTP1B was
prepared by two sequential Zeba mini centrifugal buffer exchange columns according to
manufacturer protocol. The buffer exchange columns were equilibrated before use in
sodium acetate (100 mM), Tris (50 mM), Bis-tris (50 mM, pH 7), DTPA (10 mM), and
Tween 80 (0.05%). The thiol-free enzyme was further diluted, if necessary, with sodium
acetate (100 mM), Tris (50 mM), Bis-tris (50 mM, pH 7), DTPA (10 mM), and Tween 80
(0.05%) to achieve final stock concentrations of 600 nM–8 µM active enzyme.
S3
PTP1B 1-321 and 1-298 constructs. For the crystallographic studies, PTP1B
catalytic domain constructs having cleavable polyhistidine tags were engineered. The
coding sequence for residues 1-321 was subcloned into pKA8H vector using NdeI and
BamHI site such that the N-terminal His8 tag was cleavable using tobacco etch virus
protease (TEVP). Briefly, the plasmid containing PTP1B coding sequence was amplified
using PCR. The PCR product was gel purified using a 1% DNA agarose gel. The
purified PCR product was ligated into pZErO vector at 16 °C. The resulting ligation
product was transformed into DH5α and plated onto LB agar plates supplemented with
40 µg/mL kanamycin and the plate was incubated at 37 °C overnight. Following
incubation, four single colonies were picked and grown in LB media supplemented with
40 µg/mL kanamycin. These cultures were incubated at 37 °C and 250 rpm overnight in
an incubator-shaker and further used for plasmid preparation. The isolated plasmids were
excised with NdeI and BamHI and gel purified as described above. These inserts were
ligated into pKA8H vector (cut with NdeI/BamHI and purified) followed by
transformation into DH5α. The transformants were plated on LB agar plates
supplemented with 50µg/mL ampicillin. Four single colonies were picked again for
plasmid preparation and the clone was verified by DNA sequencing.
A shorter version of PTP1B including residues 1-298 was created by inserting a
stop codon into the aforementioned plasmid. The QuickChange kit (Stratagene) was used
for this purpose, and the clone was confirmed by DNA sequencing.
Expression and Purification of PTP1B (1-298 domain). The PTP1B (1-298)
plasmid was transformed into E. coli BL21AI cells and plated on LB Agar containing
ampicillin (50 µg/mL). The plate was incubated at 37 ºC overnight and a single colony
S4
of the transformant was picked to inoculate 10 mL starter culture made of 1% tryptone
and 0.5% yeast extract. This was incubated at 37 ºC with constant shaking at 250 rpm,
overnight. The starter culture was used to inoculate 1 L of auto-induction media,3 and the
cells were allowed to shake constantly for two hours at 37 ºC and 250 rpm. After two
hours of cell growth, the temperature was reduced to 25 ºC, and 0.2 % arabinose was
added to the media. Cells were harvested after 20-21 hours by centrifugation at 4 ºC and
3500 rpm and resuspended in Buffer A (20 mM Tris, 150 mM NaCl, 10% glycerol pH
7.5). The cell pellet was quick-frozen into liquid nitrogen for later use.
Frozen cells were thawed at 4 ºC in the presence of the following protease
inhibitors: 10 µM leupeptin, 1 µM pepstatin A, 1 mM PMSF. Cells were stirred for 15-
20 minutes at 4 ºC followed by disruption using sonication. Unbroken cells and debris
were removed by centrifugation for 60 min at 17,000 rpm. The supernatant was collected
and subjected to a second centrifugation step for 30 min at 17,000 rpm. The resulting
supernatant was used for further purification by immobilized metal-ion affinity
chromatography (Ni2+-charged HiTRAP; GE Healthcare). The fractions were eluted
using buffer B (Buffer A supplemented with 1 M imidazole). Fractions containing
PTP1B were pooled and mixed with TEVP (1 mg of TEVP per 40 mg of PTP1B) and 1
mM THP. The sample was incubated for 8 h at 20 ºC and then dialyzed against buffer A.
The dialyzed protein was again loaded onto the Ni2+ charged column using buffer A.
Tag-free PTP1B was collected in both the flow-through and by elution in 3% buffer B.
The purified protein was dialyzed into 10 mM Tris, 25 mM NaCl, 1 mM EDTA, 1 mM
THP pH 7.5. Finally, the protein was distributed into thin-walled PCR tubes, quick-
frozen in liquid nitrogen, and stored at –80 ºC.
S5
SHP2 expression and purification. The SHP2 encoding plasmid was transformed
in to BL21AI cells and plated on LB–Agar supplemented with kanamycin (40 µg/ml).
The plate was incubated at 37 ˚C overnight and a single colony of the transformant was
picked to inoculate a 10 mL starter culture made of 1% tryptone and 0.5% yeast extract.
This was incubated at 37 ºC with constant shaking at 250 rpm, overnight. The starter
culture was used to inoculate 1 L of autoinduction3 media and cells were allowed to
shake constantly for 2 h at 37 ˚C and 250 rpm. After 2 h of cell growth, the temperature
was reduced to 18 ˚C and 0.2 % arabinose was added to the media. Cells were harvested
after 28 h at 4 ˚C and 3500 rpm by centrifugation and resuspended in 10 mM HEPES,
250 mM NaCl pH 7.5. The cell pellet was flash frozen in liquid nitrogen and was stored
at –80 ˚C.
Frozen cells were thawed at 4 ºC and ruptured using a sonicator. Unbroken cells
and debris was removed by centrifugation for 60 min at 17,000 rpm. The supernatant
was collected and subjected to a second centrifugation step for 30 min at 17,000 rpm.
The resulting supernatant was used for further purification by immobilized metal-ion
affinity chromatography (Ni2+-charged HiTRAP; GE Healthcare), followed by anion-
exchange chromatography (HiTRAP Q; GE Healthcare). The purified enzyme was
dialyzed into 10 mM HEPES, 250 mM NaCl, and 1 mM DTT at pH 7.5.
Supplemental Method 1. Enzyme Inactivation Assays. Thiol-free PTP1B was added
to a mixture containing various concentrations of H2O2, various concentrations of
KHCO3 and buffer to achieve final concentrations of PTP1B (0.3 µM), sodium acetate
(100 mM), Tris (50 mM), Bis-tris (50 mM, pH 7), DTPA (5 mM), and Tween 80
S6
(0.025%). After mixing, the reactions were incubated at 25 ˚C. Assays without
potassium bicarbonate employed 125-750 µM H2O2; assays containing 2 and 3.5 mM
potassium bicarbonate employed 62.5-375 µM H2O2; assays containing 7 mM potassium
bicarbonate employed 35-214 µM H2O2; assays containing 14.4 mM potassium
bicarbonate employed 19-115 µM H2O2; assays containing 25 mM potassium bicarbonate
employed 11-68 µM H2O2; assays containing 35 mM potassium bicarbonate employed 7-
46 µM H2O2; assays containing 50 mM potassium bicarbonate employed 6-37 µM H2O2.
The final pH of the inactivation mixtures did not vary more than ± 0.2 units from that of
the starting buffer. Minor variations in pH cannot account for the bicarbonate effects
reported here, as previous work shows that minor pH variations in the range 6.5-8.0 do
not substantially alter the rate at which H2O2 alone (no bicarbonate) inactivates a PTP
enzyme (see Figure 4 in ref 26). Enzyme activity remaining at various time points (30 s,
60 s, 90 s, 120 s, 150 s, and 180 s) was assessed by a method similar to those described
previously.4,5 Specifically, an aliquot (10 µL) of the inactivation reaction into an assay
mixture (490 µL) containing Bis-tris (50 mM, pH 6.0), NaCl (100 mM), DTPA (10 mM),
and p-nitrophenyl phosphate (p-NPP, 20 mM), followed by incubation at 30 ˚C for 10
min. The activity assay was quenched by addition of NaOH (500 µL of a 2 N solution in
DI water) and the amount of p-nitrophenol released during the assay determined by
measurement of the absorbance of p-nitrophenolate at 410 nm at 24 ˚C. Inactivation
reactions including additives such as tryptophan (1 mM), sodium phosphate (50 mM),
mannitol (100 mM), or desferal (1 mM) were performed as described above and the
additives were present in the inactivation mixture prior to addition of the enzyme.
S7
Inactivation assays involving SHP-2 were carried out in an identical manner except
somewhat lower enzyme concentrations were employed (~ 0.2 µM).
The value of ln(A/A0), where A is the enzyme activity remaining at time = t and
A0 is the enzyme activity at time = 0, was plotted against t and the pseudo-first-order rate
constant for inactivation at each concentration of H2O2 (kobs) calculated from the slope of
the line. The apparent second-order rate constant for the inactivation process was
obtained from the slope of a replot of kobs versus H2O2 concentration.4,6 The results of
enzyme inactivations carried out under various conditions are shown below in Figures
S1-S20. To investigate the stability of bicarbonate solutions under our reaction
conditions, we added basic BaCl2 to a mock assay solution and measured the resulting
BaCO3 precipitate.7 This analysis revealed that the solutions retained the desired
bicarbonate concentration over the course of a typical assay.
-1.9
-1.5
-1.1
-0.7
-0.3
0.10 30 60 90 120 150 180
Time (s)
ln (A
/A0)
-0.001
0.004
0.009
0.014
0.019
0.024
0 200 400 600 800H2O2 (µM)
k obs
(s-1
)
Figure S1. Inactivation of PTP1B by H2O2 alone. The lines correspond to 0, 125, 250,
375, 500, 625, 750 µM H2O2 from top to bottom. kapp = 24 ± 3 M-1 s-1.
S8
Figure S2. Inactivation of PTP1B by H2O2-KHCO3 (2 mM). The lines correspond to 0,
62, 125, 188, 250, 312, 375 µM H2O2 from top to bottom. kapp = 33 ± 5 M-1 s-1.
-4
-3
-2
-1
0
0 30 60 90 120 150 180
Time (s)
ln(A
/A0)
-0.005
0
0.005
0.01
0.015
0.02
0.025
0 100 200 300 400
H2O2 (µM)
k obs
(s-1
)
Figure S3. Inactivation of PTP1B by H2O2-KHCO3 (3.5 mM). The lines correspond to 0,
62, 125, 188, 250, 312, 375 µM H2O2 from top to bottom. kapp = 61 ± 2 M-1 s-1.
S9
-3.25
-2.5
-1.75
-1
-0.25
0 30 60 90 120 150 180
Time (s)
ln(A
/A0)
-0.002
0.002
0.006
0.01
0.014
0.018
0 50 100 150 200 250H2O2 (µM)
k obs
(s-1
)
Figure S4. Inactivation of PTP1B by H2O2-KHCO3 (7 mM). The lines correspond to 0,
36, 71, 107, 143, 179, 214 µM H2O2 from top to bottom. kapp = 74 ± 14 M-1 s-1.
-3
-2
-1
0
0 30 60 90 120 150 180
Time (s)
ln(A
/A0)
-0.002
0.004
0.01
0.016
0 20 40 60 80 100 120H2O2 (µM)
k obs
(s-1
)
Figure S5. Inactivation of PTP1B by H2O2-KHCO3 (14.4 mM). The lines correspond to
0, 19, 38, 58, 77, 96, 115 µM H2O2 top to bottom. kapp = 117 ± 11 M-1 s-1.
S10
-2.5
-2
-1.5
-1
-0.5
0
0 30 60 90 120 150 180
Time (s)
ln(A
/A0)
0
0.004
0.008
0.012
0.016
0 20 40 60 80H2O2 (µM)
k obs
(s-1
)
Figure S6. Inactivation of PTP1B by H2O2-KHCO3 (25 mM). The lines correspond to 0,
11, 23, 34, 45, 57, 68 µM H2O2 from top to bottom. kapp = 202 ± 4 M-1 s-1.
-2.5
-2
-1.5
-1
-0.5
0
0 30 60 90 120 150 180
Time (s)
ln(A
/A0)
-0.004
0
0.004
0.008
0.012
0.016
0 10 20 30 40 50
H2O2 (µM)
k obs
(s-1
)
Figure S7. Inactivation of PTP1B by H2O2-KHCO3 (35 mM). The lines correspond to 0,
8, 16, 23, 31, 39, 47 µM H2O2 from top to bottom. kapp = 274 ± 15 M-1 s-1.
S11
Figure S8. Inactivation of PTP1B by H2O2-KHCO3 (50 mM). The lines correspond to 0,
6, 12, 19, 25, 31, 38 µM H2O2 from top to bottom. kapp = 330 ± 11 M-1 s-1
Figure S9. Inactivation of PTP1B by H2O2-KHCO3 (25 mM) at 37 ˚C (rather than 25 ˚C
which was used for the assays shown above). The lines correspond to 0, 10, 21, 31, 42,
52, 62 µM H2O2 from top to bottom. kapp = 396 ± 10 M-1 s-1. At 37 ˚C, in the presence of
bicarbonate salts (NaHCO3), a slow inactivation of the enzyme occurring with an
apparent rate constant of 0.04 M-1 s-1 is observed even in the absence of H2O2.
S12
Figure S10. Preincubation of H2O2-KHCO3 (25 mM) for 2 h prior to addition of PTP1B
does not significantly change inactivation rates, relative to assays without preincubation.
The lines correspond to 0, 11, 23, 34, 45, 57, 68 µM H2O2 from top to bottom. kapp = 199
± 7 M-1 s-1 (compare to Figure S6, H2O2-KHCO3 (25 mM) without preincubation, kapp =
202 ± 4 M-1 s-1).
Figure S11. Inactivation of PTP1B by H2O2 in presence of sodium chloride (25 mM).
The lines correspond to 0, 125, 250, 375, 500, 625, 750 µM H2O2 from top to bottom.
kapp= 23 ± 4 M-1 s-1 (compare to inactivation of PTP1B by H2O2 without added NaCl,
Figure S1, kapp = 24 ± 3 M-1 s-1).
S13
Figure S12. Inactivation of PTP1B by H2O2 in presence of potassium chloride (25 mM).
The lines correspond to 0, 125, 250, 375, 500, 625, 750 µM H2O2 from top to bottom.
kapp= 25 ± 4 M-1 s-1 (compare to inactivation of PTP1B by H2O2 without added KCl,
Figure S1, kapp = 24 ± 3 M-1 s-1)
Figure S13. Inactivation of PTP1B by H2O2 in presence of magnesium chloride (2 mM).
The lines correspond to 0, 125, 250, 375, 500, 625, 750 µM H2O2 from top to bottom. kapp
= 24 ± 5 M-1s-1 (compare to inactivation of PTP1B by H2O2 without added MgCl2, Figure
S1, kapp = 24 ± 3 M-1 s-1).
S14
-1.6
-1.2
-0.8
-0.4
0
0 30 60 90 120 150 180
Time (s)
ln(A
/A0)
0
0.002
0.004
0.006
0.008
0.01
0 15 30 45 60 75H2O2 (µM)
k obs
(s-1
)
Figure S14. Inactivation of PTP1B by H2O2-KHCO3 (25 mM) in presence of the
competitive inhibitor sodium phosphate (50 mM). The lines correspond to 0, 11, 23, 34,
45, 57, 68 µM H2O2 from top to bottom. kapp = 117 ± 10 M-1 s-1 (compare to inactivation
of PTP1B by H2O2-KHCO3 (25 mM) without added sodium phosphate, Figure S6, kapp =
202 ± 4 M-1 s-1).
Figure S15. Inactivation of PTP1B (1.2 µM) by H2O2-KHCO3 (60 µM-25 mM) in the
presence and absence of the competitive inhibitor, S1 (50 nM). Enzyme activity was
measured as described. The upper points (red squares) depict the time course for the
enzyme treated with H2O2-KHCO3 (60 µM/25 mM) in the presence of S1 (50 nM). The
lower points (blue diamonds) depict the time course for the enzyme treated with H2O2-
KHCO3 (60 µM/25 mM) without S1. The IC50 of S1 against PTP1B is 47 nM.8
S15
S1
In principle, the inactivation of PTP1B by peroxides may proceed by either one-
electron or two-electron oxidation mechanisms.9,10 A one-electron process would involve
metal-mediated reduction of the peroxide bond to yield a highly reactive oxygen
radical.9,10 In the present case, a radical-mediated inactivation process seemed unlikely
given that the reactions were performed in a radical-scavenging buffer (Tris/bis-Tris) that
contained a chelator (diethylenetriamine pentaacetic acid) that inhibits metal-dependent
conversion of peroxides to oxygen-centered radicals.9 Nonetheless, we examined this
issue and found that the presence of an additional trace metal chelator, desferal (1 mM) or
radical scavengers such as mannitol (100 mM) and tryptophan (1 mM) had no significant
effect on the inactivation of PTP1B by hydrogen peroxide in the presence of bicarbonate
(Figures S16-S18). These results are consistent with the involvement of
peroxymonocarbonate in the enzyme inactivation process, as this agent has previously
been shown to act as a two-electron oxidant of sulfhydryl groups.11
S16
Figure S16. Inactivation of PTP1B by H2O2-KHCO3 (25 mM) in presence of desferal (1
mM). The lines correspond to 0, 11, 23, 34, 45, 57, 68 µM H2O2 from top to bottom. kapp
= 215 ± 14 M-1 s-1 (compare to inactivation of PTP1B by H2O2-KHCO3 (25 mM) without
added desferal, Figure S6, kapp = 202 ± 4 M-1 s-1).
Figure S17. Inactivation of PTP1B by H2O2-KHCO3 (25 mM) in presence of mannitol
(100 mM). The lines correspond to 0, 11, 23, 34, 45, 57, 68 µM H2O2 from top to bottom.
kapp= 184 ± 18 M-1 s-1 (compare to inactivation of PTP1B by H2O2-KHCO3 (25 mM)
without added mannitol, Figure S6, kapp = 202 ± 4 M-1 s-1).
S17
Figure S18. Inactivation of PTP1B by H2O2-KHCO3 (25 mM) in presence of tryptophan
(1 mM). The lines correspond to 0, 11, 23, 34, 45, 68 µM H2O2 from top to bottom. kapp
= 244 ± 23 M-1 s-1 (compare to inactivation of PTP1B by H2O2-KHCO3 (25 mM) without
added tryptophan, Figure S6, kapp = 202 ± 4 M-1 s-1).
Figure S19. Inactivation of SHP-2 by H2O2. The lines correspond to 0, 375, 500, 625,
750, 875, 1000 µM H2O2 from top to bottom. kapp= 15 ± 2 M-1 s-1.
S18
Figure S20. Inactivation of SHP-2 by H2O2 in presence of potassium bicarbonate (25
mM). The lines correspond to 0, 20, 35, 50, 65, 80, 95 µM H2O2 from top to bottom. kapp
= 167 ± 12 M-1 s-1.
Supplemental Method 2. Continuous Assay for Inactivation of PTP1B by H2O2 and
Various Bicarbonate Salts. Thiol-free PTP1B was added to a cuvette containing
substrate, buffer, bicarbonate, and H2O2 to achieve final concentrations of p-nitrophenyl
phosphate (10 mM), sodium acetate (100 mM), Bis-Tris (50 mM), Tris (50 mM),
bicarbonate salt (14.4 or 25 mM), and H2O2 (200 µM). Immediately following addition
of enzyme to the cuvette, the reaction was mixed by inversion (3x), and enzyme-
catalyzed release of p-nitrophenol was monitored at 410 nm, and 25 ºC. Data points were
taken every 2 s. The results of these assays are shown in Figure S21, below.
0
0.04
0.08
0.12
0.16
0.2
0 30 60 90 120
Time (s)
A 410
-0.04
0
0.04
0.08
0.12
0.16
0.2
0 30 60 90 120
Time (s)
A 410
S19
Figure S21. Comparison of the ability of KHCO3, NaHCO3, and NH4HCO3 to enhance
the inactivation of PTP1B by H2O2. Left side: Control enzyme (no H2O2 or HCO3–,
upper, blue line), inactivation of PTP1B by H2O2-NaHCO3 (14.4 mM, upper, teal green
curve), inactivation of PTP1B by H2O2-NH4HCO3 (14.4 mM, red curve), and inactivation
of PTP1B by H2O2-KHCO3 (14.4 mM, bottom, dark green curve). Right side: Control
enzyme (no H2O2 or HCO3–, upper, blue line), inactivation of PTP1B by H2O2-NaHCO3
(25 mM, upper, teal green curve), inactivation of PTP1B by H2O2-NH4HCO3 (25 mM,
red curve), and inactivation of PTP1B by H2O2-KHCO3 (25 mM, bottom, dark green
curve). Inactivation by H2O2-NH4HCO3 and H2O2-KHCO3 are comparable. Inactivation
by NaHCO3 is less effective, perhaps due to the lower solubility of this bicarbonate salt.
Supplemental Method 3. Gel Filtration of PTP1B Inactivated by H2O2-KHCO3.
Thiol free PTP1B (0.35 µM) was incubated for 3 min at 24 ˚C with H2O2 (70 µM) and
KHCO3 (25 mM) in a mixture containing NaOAc (100 mM), Bis-Tris (50 mM), Tris (50
mM), Surfact-Amps® 80 (0.025% v/v), DTPA (5 mM) pH 7.0. An aliquot was removed
from this solution and the remaining enzyme activity was measured as described above.
The remainder of the solution was subjected to buffer exchange via centrifugal gel
filtration using a Zeba micro spin column according to manufacturer protocol. Exchange
buffer contained: sodium acetate (100 mM), Tris-HCl (50 mM), Bis-tris (50 mM), DTPA
(10 mM), Surfact-Amps® 80 (0.05 % v/v) at pH 7. Following buffer exchange, the
resulting enzyme-containing filtrate was assayed for PTP activity as described above.
The results are shown in Figure S22, below.
S20
Figure S22. Activity of H2O2-KHCO3-inactivated PTP1B does not return following gel
filtration. The first and second bars show the activity of control, untreated enzyme before
and after gel filtration. The second two bars show the “activity” of inactivated enzyme
before and after gel filtration.
Supplemental Method 4. Dialysis of PTP1B Inactivated by H2O2-KHCO3. Thiol free
PTP1B (0.35 µM) was incubated for 3 min at 24 ˚C with H2O2 (70 µM) and KHCO3 (25
mM) in a mixture containing sodium acetate (100 mM), Bis-Tris (50 mM), Tris (50 mM),
Surfact-Amps® 80 (0.025% v/v), DTPA (5 mM) pH 7.0. An aliquot was removed from
this solution and the remaining enzyme activity was measured as described above. The
remainder of the solution was loaded onto a Slide-A-Lyzer MINI Dialysis Unit and
dialyzed for 100 min at 4 ˚C against dialysis buffer consisting of Tris (50 mM), Bis-Tris
(50 mM), NaOAc (100 mM), DTPA (10 mM), and Surfact-Amps® 80 (0.05% v/v)
detergent at pH 7.0. Following dialysis, the solution inside the dialysis chamber was
assayed for activity as described above. The results are shown in Figure S23, below.
S21
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
E beforedialysis
E afterdialysis
Bicarbbefore
dialysis
Bicarb afterdialysis
A 410
Figure S23. Activity of H2O2-KHCO3-inactivated PTP1B does not return following
dialysis. The first and second bars show the activity of control, untreated enzyme before
and after dialysis. The second two bars show the “activity” of inactivated enzyme before
and after dialysis.
Supplemental Method 5. Treatment of PTP1B Inactivated by H2O2 or H2O2-
KHCO3 with Dithiothreitol (DTT). Thiol free PTP1B (0.64 µM) was incubated for 3
min at 24 ˚C with H2O2 (375 µM) in a mixture containing NaOAc (100 mM), Bis-Tris
(50 mM), Tris (50 mM), Surfact-Amps® 80 (0.025% v/v), DTPA (5 mM) pH 7.0. An
aliquot was removed from this solution and the remaining enzyme activity was measured
as described above. To the remainder of the solution was added DTT to a final
concentration of 50 mM. The resulting mixture was incubated at 25 ˚C for 30 min and
then assayed for PTP activity as described above. The results of this assay are shown
below in Figure S24.
S22
18% activity remaining
DTT 96% returned
0
0.4
0.8
1.2
1.6
C ontrol H 2O2 C ontrol H 2O2
A41
0
DTT 99% returned
23% activity remaining
0
0.4
0.8
1.2
1.6
C ontrol B ic arb C ontrol B ic arb
A41
0
Figure S24. Treatment with DTT leads to the recovery of activity of oxidatively-
inactivated PTP1B. In each plot, the first and second bars correspond to the activity of
control, untreated enzyme and inactivated enzyme, respectively (no DTT treatment). The
second two bars show the activity of control, untreated enzyme and inactivated enzyme,
following treatment with DTT as described above.
Supplemental Method 6. Catalytic activity of SHP-2 inactivated by H2O2-KHCO3
can be recovered by treatment with dithiothreitol (DTT). Thiol-free SHP-2 (0.45
µM) was inactivated by treatment with H2O2 (150 µM) and KHCO3 (25 mM) in assay
buffer containing sodium acetate (100 mM), bis-Tris (50 mM, pH 7.0), Tris (50 mM),
DTPA (50 µM), Tween-80 (0.00025%), and p-NPP (10 mM, 1 mL final volume). The
reaction was carried out in a quartz cuvette and the enzyme activity was continuously
monitored by following the release of p-nitrophenol at 410 nm. When enzyme
inactivation was complete, as indicated by a plateau in the production of p-nitrophenol,
DTT (5 µL of a 1 M in H2O, to yield a final concentration of 5 mM DTT) was added to
the cuvette using a long pipette tip, the solution mixed by pumping the pipette three
S23
times, and the recovery of enzyme activity observed by monitoring the absorbance
increase at 410 nm. The results are shown below in Figure S25, below.
Figure S25. Catalytic activity of SHP-2 inactivated by H2O2-KHCO3 can be recovered
by treatment with dithiothreitol (DTT).
0
0.05
0.1
0.15
0.2
0.25
0.3
0 50 100 150 200
T ime (s )
A41
0
Figure S26. Inactivation of SHP-2 by H2O2-KHCO3 (430 µM/25 mM) in presence of the
competitive inhibitor sodium phosphate (50 mM). Enzyme activity was measured as
described above in Supplemental Methods 1. The upper points (blue diamonds) depict
the loss of activity in enzyme treated with H2O2-KHCO3 (430 µM/25 mM) in the
presence of sodium phosphate (50 mM). The lower points (red squares) depict the loss of
activity in enzyme treated with H2O2-KHCO3 (430 µM/25 mM) without sodium
phosphate.
Supplemental Method 7. Inactivation of the Cysteine Protease Papain by H2O2 and
H2O2-KHCO3. Papain (10 mg/mL) was activated by incubation with DTT (2 mM) for
S24
30 min at 25 ˚C in sodium phosphate buffer (50 mM, pH 7) containing EDTA (2.5 mM)
as described previously.12 Thiol was removed from the activated enzyme as described for
PTP1B in the Materials Section. The buffer exchange columns were equilibrated with
sodium phosphate (50 mM, pH 7), EDTA (2.5 mM). The resulting “thiol-free” papain
was used immediately in inactivation assays. Typical inactivation assays contained
various concentrations of H2O2 either with, or without, KHCO3 (25 mM) along with
papain (1 mg/mL), sodium phosphate (50 mM, pH 7), EDTA (2.5 mM). At various times
an aliquot (100 µL) of the inactivation reaction was transferred into a cuvette containing
substrate solution (900 µL) to give final concentrations of Nα-benzoyl-L-arginine 4-
nitroanilide hydrochloride (BAPNA,13 1 mM), sodium phosphate (45 mM), EDTA (2.25
mM), and DMSO (10% v/v). Enzyme activity was measured by monitoring the initial
rate of release of 4-nitroaniline at 410 nm (first 30 s). The percent remaining activity was
calculated based on comparison to a control assay containing no H2O2 or KHCO3. The
results are shown below in Figures S27 and S28.
Figure S27. Inactivation of Papain by H2O2 alone. The lines correspond to 0, 10, 20, 40,
60, 80 µM H2O2 from top to bottom. kapp = 43 ± 7 M-1 s-1. This value is comparable to
S25
published literature values for the inactivation of cysteine proteases by H2O2 in the
absence of substrate.14,15
-2.5
-2
-1.5
-1
-0.5
00 180 360 540 720 900
Time (s)
ln (A
/A0)
0
0.001
0.002
0.003
0 10 20 30
H2O2 (µM)
k obs
(s-1
)
Figure S28. Inactivation of Papain by H2O2-KHCO3 (25 mM). The lines correspond to 0,
10, 15, 20, 30 µM H2O2 from top to bottom. kapp = 83 ± 9 M-1 s-1.
Supplemental Methods 8. Crystal structure determination. Crystallization trials
were performed at 4º C using sitting drop vapor diffusion and a 10 mg/mL stock solution
of PTP1B (1-298). Diffraction quality crystals were grown using 11-18 % PEG3000, 0.1
M HEPES pH 7.0-8.0, 0.2 M magnesium acetate, and 2 mM TCEP as reported
previously.16
Crystals of inactivated PTP1B were prepared by soaking PTP1B crystals at room
temperature. The crystals were first cryoprotected using 20% PEG3000, 0.1 M HEPES
pH 7.0, 0.2 M magnesium acetate, and 20% PEG 200, and then transferred to a solution
containing the cryobuffer supplemented with 25 mM KHCO3 and 50 µM H2O2. The soak
time was varied from 20 to 45 min. Crystals were picked up with Hampton loops and
plunged into liquid nitrogen.
A 1.7 Å resolution X-ray diffraction data set was collected at ALS beamline 4.2.2
S26
and processed using d*TREK (Table S1).17 The crystals have space group P3121 with
unit cell dimensions of a = 88.4 Å and c = 104.3 Å; there is 1 molecule in the asymmetric
unit. Molecular replacement phasing as implemented in PHENIX AutoMR was used.18
The search model was derived from a native PTP1B structure (PDB code 2f71)19 with
following residues omitted: 46-49, 180-188, and 215-221. Note that this list includes the
active site Cys215 and flexible active site loops whose conformations are sensitive to the
oxidation state of Cys215 and the presence of bound ligands. The model from molecular
replacement was improved with iterative rounds of model building in Coot20 and
refinement in PHENIX.21
The 1.7 Å resolution electron density map clearly showed the formation of the
cyclic sulfenyl amide involving Cys215 and Ser216 (Figure S29). The conformations of
the P-loop and other flexible active site loops are identical to those described previously
for crystals soaked in H2O2.22,23 For example, electron density for the P-loop is shown in
Figure S29.
S27
Table S1. X-ray data collection and refinement statisticsa
Wavelength (Å) 1. 0000 Data collection resolution (Å) 44.21 - 1.70 (1.76 - 1.70) No. of Observations
571812
No. of unique reflections 52290 Rmerge(I) 0.063 (0.552) Average I/σ 12.6 (2.8) Completeness (%) 100.0 (100.0) Redundancy 10.94 (11.00) Refinement resolution (Å) 44.21 - 1.70 (1.76 - 1.70) Rcryst 0.197 (0.290) Rfree
b 0.210 (0.303) No. of protein residues 282 No. of protein atoms 2265 No. of water molecules 170 Average B-factor (Å2) Protein 32.1 Water 38.0 Mg+2 35.0 rmsdc Bonds (Å) 0.006 Angles (deg) 1.051 Ramachandran plotd Favored (%) 98.21 Allowed (%) 1.79
Outliers (%) 0.00 Coordinate error (Å)e 0.21 PDB code 3SME aValues for the outer resolution shell of data are given in parenthesis. b5 % random test set. cCompared to the parameters of Engh and Huber.24 dThe Ramachandran plot was generated with RAMPAGE.25 eMaximum likelihood-based coordinate error estimate.
S28
Figure S29. Stereographic view of PTP inactivated by 25 mM KHCO3 and 50 µM H2O2
(gray) or H2O2 alone (yellow, PDB code 1OEM). The P-loop is shown in sticks (residues
214-221). The cage represents a simulated annealing σA-weighted Fo-Fc omit map
contoured at 3.0σ. Prior to map calculation, the P-loop was omitted and simulated
annealing refinement was performed using PHENIX.
Acknowledgement. We thank Dr. Ernest Asante-Appiah (Merck) for providing the
PTP1B inhibitor S1.
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Supplemental Literature Citations
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