the biological buffer bicarbonate/co potentiates h o...

Published: September 13, 2011

r 2011 American Chemical Society 15803 dx.doi.org/10.1021/ja2077137 | J. Am. Chem. Soc. 2011, 133, 15803–15805

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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 [email protected]; [email protected]

’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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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: [email protected] and [email protected], 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,


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

)



-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

)



-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

)



S11


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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