Combinatorial Chemical Reengineering of the Alpha Class

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Bioconjugate Chem. 2004, 15, 718−727

Combinatorial Chemical Reengineering of the Alpha Class Glutathione Transferases Johan Viljanen, Lotta Tegler, and Kerstin S. Broo* IFM, Department of Organic Chemistry, Linko¨ping University, S-581 83 Linko¨ping, Sweden. Received October 21, 2003; Revised Manuscript Received March 16, 2004

Previously, we discovered that human glutathione transferases (hGSTs) from the alpha class can be rapidly and quantitatively modified on a single tyrosine residue (Y9) using thioesters of glutathione (GS-thioesters) as acylating reagents. The current work was aimed at exploring the potential of this site-directed acylation using a combinatorial approach, and for this purpose a panel of 17 GS-thioesters were synthesized in parallel and used in screening experiments with the isoforms hGSTs A1-1, A2-2, A3-3, and A4-4. Through analytical HPLC and MALDI-MS experiments, we found that between 70 and 80% of the reagents are accepted and this is thus a very versatile reaction. The range of ligands that can be used to covalently reprogram these proteins is now expanded to include functionalities such as fluorescent groups, a photochemical probe, and an aldehyde as a handle for further chemical derivatization. This site-specific modification reaction thus allows us to create novel functional proteins with a great variety of artificial chemical groups in order to, for example, specifically tag GSTs in biological samples or create novel enzymatic function using appropriate GS-thioesters.

Creating new function in an existing protein scaffold is a challenging task, even though progress has been made lately through rational design (1-3) and combinatorial approaches (4, 5). A less explored route to novel functional proteins is that of chemical modification of a protein scaffold to introduce an artificial group (6-9). Picking a versatile scaffold is of course crucial in any protein reengineering project, and we have focused on the glutathione transferases (GSTs, EC 2.5.1.18),1,2 a large family of phase II detoxication enzymes that catalyze the nucleophilic addition of glutathione (GSH, Scheme 1) to a broad range of hydrophobic electrophilic molcules (10-13). The proteins exist as homo- or heterodimers (12, 14), and the mode of binding of GSH in the N-terminal domain is essentially conserved throughout the classes (10, 15). The differences between the isoenzymes are generally located in the promiscuous hydrophobic electrophile binding sites (H-sites) to provide a broad substrate specificity (16). The catalytic function is highly dependent on a conserved tyrosine, serine or cysteine residue in the glutathione-binding site (G-site) * To whom correspondence should be addressed. Fax +4613-12 25 87, e-mail: [email protected]. 1 Abbreviations: ACN, acetonitrile; C18, octadecyl; DCM, dichloromethane; DIPCDI, N,N′-diisopropylcarbodiimide; DIPEA, diisopropylethylamine; DMF, dimethylformamide; DTNB, 5, 5′-dithiobisnitrobenzoic acid; Fmoc, 9-fluorenylmethyloxycarbonyl; GSH, glutathione, γ-Glu-Cys-Gly; GST, glutathione transferase; hGST A1-1, GST A1-1 isoform from human; G-site, glutathione-binding site; GS-thioester, thioester of glutathione; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; H-site, hydrophobic electrophile binding site; NMR, nuclear magnetic resonance; MALDI-MS, matrix assisted laser desorption mass spectrometry; Mmt, 4-methoxytrityl; NaPi, sodium phosphate; TFA, trifluoroacetic acid; TNB, thionitrobenzoic acid; TOF, time-of-flight; UV, ultraviolet. 2 This nomenclature is derived from that recommended by Mannervik, B. et al. (1992) Biochem J 282 (Pt 1), 305-6.

Scheme 1

that aids in the ionization of GSH to form the more reactive thiolate ion (10). The modular features of these proteins with a stringent G-site and an adjacent, promiscuous H-site, in combination with their stability and ease of purification (17) and the vast knowledge of structure-activity relationships (18), are all factors that point to the GSTs as ideal candidates in protein engineering experiments (19-26) with the goal of obtaining novel function. Previously, we found that a thioester of glutathione (GS-thioester) termed GSB (Scheme 1) was able to rapidly modify Y9 (Figure 1) of the human GSTs from the alpha class (27). The reaction is highly specific and, for example, targets one out of 51 nucleophiles in hGST A1-1. Out of the three GS-thioesters we investigated, two were able to acylate the alpha class GSTs (27). The covalent modification reaction presents us with the

10.1021/bc034192+ CCC: $27.50 © 2004 American Chemical Society Published on Web 05/21/2004

Expanding the Range of GST Acylation Reagents

Bioconjugate Chem., Vol. 15, No. 4, 2004 719

Figure 1. A close-up of the crystal structure (1GUH) of hGST A1-1 in a complex with S-benzyl-glutathione (15). The distance between the hydroxyl oxygen of Y9 and the ligand benzyl CH2 is 4.2 Å. The distinguishing feature of the alpha class GSTs helix 9 (R9) is also indicated in the figure. The protein is dimeric but shown here as a monomer for reasons of clarity of presentation. Chart 1

opportunity to create novel functional proteins through the design and synthesis of GS-thioesters with appropriate chemical groups. Thioesters could be synthesized to introduce, for example, a fluorescent group that could bind in the nearby H-site to form the basis for a biosensor system, or an artificial catalytic group that could result in a new type of enzymatic activity (28, 29). It is thus important to understand the potential and limitations of the reaction. To do so, we synthesized 17 different GSthioesters (Chart 1) in parallel and screened this collection against hGSTs A1-1, A2-2, A3-3, and A4-4. The

combined screening of both reagents and proteins was done in order to investigate if any of the alpha class isoenzymes is better suited as a scaffold for protein reengineering experiments and also to try to obtain general information about reagent requirements. The GSthioesters therefore span a wide range of reactivities, hydrophobicities, shapes, and sizes. Some of the reagents have functionalities that can be used in downstream experiments, for example fluorescent groups (GS-3, GS4) and a handle for further chemical derivatization (GS15).

720 Bioconjugate Chem., Vol. 15, No. 4, 2004 MATERIALS AND METHODS

Protected amino acids and acids (DiversiChem kit) were purchased from Novabiochem (La¨ufelfingen, Switzerland). Preactivated acids (5-carboxyfluorescein succinimidyl ester, p-formylbenzoic acid succinimidyl ester, 4-benzoylbenzoic acid succinimidyl ester) were purchased from Molcular Probes. All chemicals and reagents used were of the highest purity available. The glutathione transferases were kind gifts from the group of Professor Bengt Mannervik at the Department of Biochemistry, Uppsala University, Sweden. All solid-phase syntheses were carried out in a VacMaster (International Sorbent Technology). The HPLC experiments were carried out using a Kromasil C8-column (4.6 mm × 250 mm, Supelco inc.) attached to a Varian system with a ProStar 230 Delivery System, a ProStar 330 Photodiode Array Detector, controlled by the Varian LC Software. All mass spectra were recorded with a MALDI/TOF-MS (Voyager System 4212, Applied Biosystems) with detection in the positive mode. The UV measurements were performed using a Varian Cay 100 Scan UV-visible spectrophotometer and analyzed with the CaryWin UV software. Synthesis of Fully Protected, Polymer-Bound, γ-Glu-Cys-Gly. The tripeptide γ-Glu-Cys-Gly (0.71 mmol scale) was synthesized using standard Fmoc protocols starting from Fmoc-Gly-Wang resin. The thiol was orthogonally protected with the Mmt group to enable selective deprotection with 1% TFA in DCM. Each deprotection and coupling step was monitored by a Kaiser test (30), to estimate the amount of free amino groups. After completed synthesis, the polymer was washed thoroughly with DMF and DCM and dried under vacuum. Synthesis of Thioester Reagents. The GS-thioesters were synthesized in parallel using 0.03 mmol polymer for each ligand. In each case, the Mmt-group was removed with DCM:TFA:TIS (97:1:2). After deprotection of the thiol the polymer was washed with DMF, and the subsequent coupling of the acid was performed in DMF using acid: HOBt:DIPCDI:DIPEA (2:3:3:6) with addition of a catalytic amount of 4-pyrrolidinopyridine. The couplings were allowed to proceed overnight following an additional boosting of the same amount of couplingcocktail after 1 h. Preactivated acids (5-carboxyfluorescein succinimidyl ester, p-formylbenzoic acid succinimidyl ester, 4-benzoylbenzoic acid succinimidyl ester) were coupled using a mixture of ester:DIPEA (2:6) in DMF during the first hour, after which DIPCIDI:HOBt:DIPEA (2:3:3:6) was added and the coupling was allowed to proceed overnight. Global deprotection and simultaneous cleavage from the solid support was achieved with TFA: H2O (97.5:2.5). Following precipitation with ice-cold diethyl ether and lyophilization, each GS-thioester was dissolved in 5 mL of Milli-Q water as a stock solution used for all further experiments. The concentrations of the stock solutions were calculated from the weights of the products assuming 100% purity. The yields of the syntheses, according to weighing, range from 11% to 48%, with most of them around 30%. The peptides were analyzed with reversed-phase HPLC using a C8 column and a shallow H2O:ACN (both 0.1% TFA) gradient. The identities of the products were confirmed with MALDITOF MS using an R-cyano-4-hydroxycinnamic acid matrix with detection in the positive mode. The measured masses of the GS-thioesters were in good agreement with the calculated masses. Amount of GSH in the GS-Thioester Stock Solutions. The Ellmans test (31) was carried out to analyze the amount of unreacted GSH in the GS-thioester solu-

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tions. The samples were prepared by adding 10 µL of GSthioester stock solution to 90 µL of 10 mM DTNB in 0.1 M NaPi, pH 8. Thiol groups react with DTNB and produces equivalent amounts of TNB. The amount of produced TNB (412 ) 13 600 M-1 cm-1) was measured using a Fluostar Galaxy plate-reader (BMG Lab technologies) equipped with a 420 nm filter. The amount of free GSH varied from 0.5 to 3%. These calculations did not take into account the GS-thioesters intrinsic absorbances at this wavelength and correspond therefore to an upper limit. Glutathione Transferases. The activities of the GSTs were checked with a standard assay (32) containing 1 mM each of 1-chloro-2,4-dinitrobenzene and GSH (pH 6.5 in 0.1 M NaPi at 30 °C). Protein concentrations were estimated using previously published extinction coefficients for hGSTs A1-1 (33), A2-2 (34), A3-3 (35), A4-4 (23). The proteins were stored in NaPi-buffered solutions containing NaCl and 10% glycerol at -80 °C or -20 °C until used in the experiments. Modification Reactions. A typical modification reaction was performed with 100 µM GS-thioester and 50 µM GST in buffer (0.1 M NaPi, pH 7). The reaction was allowed to proceed at 25 °C for 5 h. To control the temperature, the incubation was performed in an Eppendorf Master cycler gradient. Proteolytic Digestion of Modified GSTs. The digestions were performed in the following way: 5 µL of modification mixture was diluted with 13 µL of buffer (50 mM NH4OAc, pH 4), 2 µL of Staphylococcus areus V-8 Protease from Pierce Biotechnology, Inc., Rockford, IL, (stock solution 1 µg/µL) was added, and the sample was incubated at 25 °C for up to 2 h. The sample was then acidified with 20 µL of 0.1% TFA and desalted with C18 ZipTips (Millipore Corporation, Bedford, MA). In the case of tryptic digestion, 10 µL of modification mixture was diluted with 10 µL of buffer (0.2 M NaHCO3, pH 8), and the reaction was initiated by adding 1 µL of trypsin (stock solution 0.2 µg/µL in 0.1 mM HCl) and allowed to proceed at 25 °C for up to 2 h. The sample was then analyzed directly or acidified and desalted (C18 ZipTips) prior to MALDI-MS analysis. HPLC Analysis of the Modification Reactions of hGST A1-1. Each GS-thioester (100 µM) was incubated with hGST A1-1 (50 µM) at 25 °C (in 100 mM NaPi, pH 7) in a total volume of 36 µL. After 5 h, the reaction was quenched by adding 4 µL of a mixture of TFA (final concentration 0.4% v:v) and internal standard, 2-chloro4-nitrophenol (final concentration 200 µM). As a reference, each GS-thioester was also incubated in buffer without protein and quenched after 5 h. All samples were then analyzed with reversed-phase HPLC using a C8 column and a shallow H2O:ACN (both 0.1% TFA) gradient. The injection volumes were 25 µL. The quenched samples from the HPLC experiments were also digested with trypsin as described before except that 2 µL of each sample mixture was diluted with 18 µL of buffer before adding the enzyme. After 2 h of incubation, the samples were analyzed with MALDI-MS. To each sample was added 0.1 M citrate to the matrix on the MALDI plate to enable analysis without desalting. In some cases, the peak of the modified protein fragment coincided with other peptides, and we then used S. areus V-8 protease to digest the protein mixture. Acyl Group Exchange Experiment. hGST A1-1 (5 µM) was reacted overnight at 25 °C with 100 µM GSB. The reaction mixture was desalted with YM-30 filters (Millipore) and washed with 0.1 mM NaPi pH 7. The sample was then diluted back to the original volume and



Figure 2. The reaction between GS-2 (100 µM) and hGST A1-1 (50 µM) at pH 7 monitored by (A) HPLC and (B) MALDI-MS. The chromatograms are shown at 250 nm for naphthoic acid, a reference sample of GS-2 incubated without protein, and the GS-2 sample incubated with protein for 5 h at 25 °C. The internal standard (2-chloro-4-nitrophenol) is abbreviated IS. The MALDI-MS are recorded after proteolytic (S. aureus V8) digestion, and both the unmodified and the modified fragment (AEKPKLHY*FNARGRME) containing Y9 are indicated in the sample incubated with GS-2.

subjected to 100 or 10 µM GS-2. Samples were withdrawn at 15 min, 1 h, 5 h, and after overnight incubation at 25 °C. The time-points were digested with trypsin and analyzed by MALDI-MS.

Table 1. GS-thioesters after Five Hours Incubation, pH 7 and 25 °C HPLCa name

RESULTS

Quality and Yield of GS-thioesters. The GSthioesters were synthesized from one batch of fully protected, polymer-bound GSH with yields ranging from 11% to 48% according to weighing. All GS-thioesters were identified with MALDI-MS. According to the Ellmans test (31), the amount of free thiol, i.e., free GSH, was between 0,5% and 3%. In addition to the Ellmans tests, the qualities of the products were evaluated from HPLC (for an example, see Figure 2, GS-2) and deemed satisfactory for use in the screening experiments without further purification. Addition of the GS-thioester Panel to hGST A11. The outcome of adding the GS-thioesters to hGST A1-1 was investigated by analytical HPLC. Each reagent (100 µM, except GS-17, see Discussion) was incubated with protein (50 µM) at 25 °C, and the reaction was quenched after 5 h by adding a solution of TFA (0.4% final concentration) and an internal standard (2-chloro-4nitrophenol). The thoroughly investigated GSB (27) was also incorporated as one of the reagents in order to include a positive control. As a reference, each GSthioester was also incubated without protein for 5 h in buffer at 25 °C. The samples were then analyzed with reversed-phase HPLC (Figure 2). The peak area of each reagent incubated with protein was compared with the peak area of the reagent in the reference sample and also to a sample of the corresponding acid injected on the same gradient. These data were used to assess whether there was any decrease in thioester concentration and/ or increase in free acid concentration (Table 1). The quenched samples from the HPLC experiments were also analyzed by MALDI-MS following proteolytic digestion in order to ensure that decreases in peak areas corresponded to modifications of Y9 (Figure 2). If Y9 becomes covalently modified, the mass of the fragment containing Y9 increases with the mass of the corresponding acid minus the weight of water (Scheme 1). Out of

GSB GS-1 GS-2 GS-3 GS-4 GS-5 GS-6 GS-7 GS-8 GS-9 GS-10 GS-11 GS-12 GS-13 GS-14 GS-15 GS-16 GS-17

hGSTA1-1 sample acid reference sample GSX peak peak acid peak MALDI-MSb + + + + + + + + + + + + + + +

+ + + -

+ -

+ + + + + + + + + + + + + +

a + corresponds to decrease of GSX peak (cutoff 20% decrease) and - corresponds to appearance of acid peak (cutoff 5% increase). b + and - corresponds to modified or not modified Y9 fragment, respectively.

the 18 GS-thioesters we investigated (GSB included), 14, or 78%, were able to covalently modify hGST-A1 as judged by both HPLC and MALDI-MS (Table 1). MALDI-MS Screening of the GS-thioester Panel against hGSTs A2-2, A3-3, and A4-4. The acyl group preferences of hGSTs A2-2, A3-3, and A4-4 were investigated using the panel of GS-thioesters with GSB included as a positive control. For each reagent, the reaction was monitored with a GS-thioester concentration of 100 µM (except GS-17, see Discussion), and protein concentrations ranging from 5 µM to 50 µM, with an incubation time of 5 h at 25 °C. After incubation, the samples were digested with trypsin or S. aureus protease V-8 depending on possible overlaps from other fragments. Through MALDI-MS experiments, we then identified modified Y9-fragments for 83% of the GS-thioesters for both hGSTs A2-2 and A3-3, and for 72% of the reagents for hGST A4-4 (Table 2, Figure 3). A positive identifica-

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Table 2. Modification Reactions of the Alpha Class GSTs

Chart 2

hGST substrate

A1-1

A2-2

A3-3

A4-4

GSB GS-1 GS-2 GS-3 GS-4 GS-5 GS-6 GS-7 GS-8 GS-9 GS-10 GS-11 GS-12 GS-13 GS-14 GS-15 GS-16 GS-17

+ + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + +

(a) + and - corresponds to modified or not modified Y9 fragment.

tion corresponds to more than 10% modified Y9-fragment relative to the parent fragment (unmodified Y9). This relatively low threshold was set because MALDI-MS is not a quantitative technique and also because of the relatively large error in estimating GS-thioester concentrations (Discussion). Exchange of Acyl Groups in Already Modified hGST A1-1. The ability to exchange acyl groups in already modified hGST A1-1 was investigated by subjecting benzoic acid-modified hGST A1-1 (5 µM) to GS-2 at two different concentrations (10 and 100 µM) (Figure 4). Time points were withdrawn, and the reaction mixtures were digested and analyzed with MALD-MS to obtain semiquantitative data regarding the amount of modification for each acyl group. DISCUSSION

The speed and selectivity of the previously described site-directed covalent modification reaction between hGSTs from the alpha class and GS-thioesters (27) encouraged

us to investigate the potential of this reaction both with respect to protein promiscuity and also to the linked question of reagent requirements. The natural substrates of GSTs differ widely in size and hydrophobicity (16), and we therefore found it likely that this would be a quite general modification reaction. The previous report was based on a set of three different GS-thioesters (Scheme 1 and Chart 2); GSB, GS-ANT, and S-lactoylglutathione. Out of these, the only one that did not react with Y9 was the only one that was not an aromatic GS-thioester (Slactoylglutathione). This, in combination with the report that rat GST A1-1, highly homologous to hGST A1-1, hydrolyses a nonaromatic GS-thioester (36), raised the question that this perhaps was a unique reaction only involving aromatic GS-thioesters. With the intention to study this, we therefore included GS-16 and GS-17 (Scheme 1) in the panel of GS-thioesters. The individual GS-thioesters within the set of 17 new reagents thus vary widely in size, shape, hydrophobicity, and also reactivity (pKa of acid) in order to screen as many parameters as possible. HPLC and MALDI-MS Analysis of the Reactions between the GS-thioester Panel and hGST A1-1. The outcome of adding the panel of reagents to hGST A1-1 was analyzed using both HPLC and MALDI-MS with the objective to get detailed information about the fate of the acyl group. Previously, we did a thorough investigation and could conclude that GSB reacts with hGST A1-1 in a stoichiometric fashion to produce 1 equiv of GSH and one 1 equiv of benzoic acid-modified protein (27). The reagent GSB is not hydrolyzed upon incubation at pH 7, even after 9 days, but instead the acyl group migrates to the R-amino group of GSH. However, we could not assume that this was valid for all GS-thioesters. To track the acyl group, we therefore incubated the reagents with

Figure 3. MALDI-MS spectra of proteolytic peptide fragments from hGSTs (A) A1-1, (B) A2-2, (C) A3-3, and (D) A4-4 following 5 h incubations with different GS-thioesters (100 µM) specified in the figure. The interval containing the Y9 fragments are shown in the figure. Parent fragments are indicated with asterisks, and modified fragments are highlighted with arrows.



Figure 4. Benzoic acid-modified hGST A1-1 (5 µM) was subjected to GS-2, time points were withdrawn, and the reaction mixtures were digested with trypsin and analyzed with MALDI-MS. The figure shows (A) the spectrum before GS-2 was added and (B) the spectrum after 5 h incubation with 100 µM GS-2. The interval containing the Y9 fragments are shown in the figure. The parent fragment is indicated with an asterisk.

and without hGST A1-1 for 5 h, and we also injected the acids using the same gradient. A decrease in the peak area of the GS-thioester reagent that is due to hydrolysis can then be identified in the chromatogram. If a rearrangement reaction takes place, the result is not so easily interpreted since a migration event, in combination with oxidation of the produced thiol, would result in a total of up to five or even seven peaks, should hydrolysis also occur. The areas of the peaks thereby become very low and would be hard to detect at the concentration of GSthioesters used during the experiments. Because of the relative concentration of reagent to protein (2:1), the maximum decrease of any GS-thioester peak should be 50% when hGST A1-1 is added if the event corresponds to modification of hGST A1-1. However, since the concentrations of the GS-thioester solutions are derived from weighing, the decreases could be greater than 50% even if the only event that takes place is modification of the protein in the sample. This is due to several reasons: this is a screening approach and we have not purified each substrate but used them after only ether precipitation (see above), the mixtures contain a small amount of free GSH (less than 3%), and peptides are also inherently hygroscopic. Weighing does therefore overestimate the amount of thioester reagent in the reaction mixture. The HPLC data is therefore presented in Table 1 as decrease or no decrease of thioester reagent. The exact figures can be found in the Supporting Information. The conditions we have used to analyze these reactions are not suitable

for direct detection of the modified protein since it elutes as a very broad peak preceding the internal standard (Figure 2). Through these experiments we found that the great majority of the reagents are stable under the reaction conditions (5 h incubation at 25 °C). The migration event could be observed for GS-17 to some extent, and this, in combination with the poor extinction coefficient, motivated us to use a higher initial concentration of this reagent (250 µM) in the derivatization experiments. Only in one case (GS-11) was an acid peak observed in the absence of protein. The quenched samples from the HPLC experiments were also digested and analyzed directly with MALDIMS, and the results from the HPLC experiments could be verified (Table 1) in that we detected fragments that corresponded to modified Y9. In the presence of hGST A1-1, some of the GS-thioesters were hydrolyzed to some extent, most notably GS-11 and GS-16; however, none of these did show up as modified Y9 fragments in the MALDI-MS experiments and were thus classified accordingly. In one case (GS-12) we could observe a small peak of the expected mass in the beginning of the ionization process, but the peak disappeared as the laser beam continued, and it did not reappear. It is perhaps likely that this is due to the acyl group itself, since it is photolabile and it might thus be destroyed in the MALDIMS. Overall, the correlation between the HPLC and the MALDI-MS results is very good. The combined results


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Figure 5. Close-ups of the crystal structures of hGST A1-1 (1GUH) (15) and hGST A4-4 (1GUL) (41) crystallized with glutathione conjugates showing the potential “back-door” leading out from the hydrophobic binding site. The H-site residues are shown in CPK, the G-site residues Y9 in ball-and-stick, and the glutathione-derived ligands in line representation. Both proteins are dimeric, but here are shown as monomers for reasons of clarity of presentation.

from MALDI-MS and HPLC thus showed that 14 of the GS-thioesters, or 78%, were able to modify hGST A1-1 (Table 1). Modification of the Alpha Class hGSTs A2-2, A33, and A4-4. To explore the versatility of each of the alpha class proteins, we also screened hGSTs A2-2, A33, and A4-4 against all GS-thioesters (Table 2) using proteolytic digestion and MALDI-MS. Since the HPLC experiments had demonstrated that the GS-thioesters were stable in the absence of hGST A1-1, we chose to focus on the MALDI-MS experiments that more directly show the presence of modified protein. We found that all of the alpha class GSTs are highly promiscuous, and the percentage of accepted modification reagents range from 72 to 83%. This is perhaps not so surprising since the isoenzymes catalyze reactions where the hydrophobic substrates vary hugely in size and hydrophobicity (16, 21, 34, 37-40). The relaxed hydrophobic substrate specificity of the GSTs that originates from their biological function as detoxication enzymes make them ideally suited as protein scaffolds in reprogramming experiments, and there is also room left, both in the G-site and in the H-site, even after Y9 has been modified with a new functional group. In all, the alpha class GSTs seems to be equally well suited as protein scaffolds in chemical reengineering experiments since they can all be reprogrammed with a large variety of functional groups. The extent of modification varies, which is not surprising, since we used standardized conditions with respect to incubation time, temperature, and reagent concentration for all reactions. In our previous paper (27), we could observe that the extent of modification using GSB and hGST A1-1 was a function of time and concentration of reagent. We tried to maximize the extent of modification in one case, hGST A3-3 and GS-15 (Figure 3C), but we were not able to achieve more than a slight increase of modified protein even after raising the concentration of modifying reagent and allowing 24 h of modification. It is probably due to hydrolysis of the reagent as can be seen in Table 1 already after 5 h and also perhaps due to the slow deterioration of the protein at room temperature. The rate of modification, hydrolysis, and deterioration are in this case probably incompatible. There are crystal structures of hGSTs A1-1 (15) and A4-4 (41) (Figure 5), and the distinguishing feature of the alpha class is helix 9 (R9) that forms a lid over the

active site (Figure 1). The H-sites of the alpha class proteins differ from hGSTs A1-1 in three (A2-2), four (A33), and nine amino acids (A4-4), respectively (Figure 6). The differences within the alpha class lead to subtle differences in substrate specificity and also in the dynamic features of the proteins. For instance, R9 can be observed in the crystal structure of hGST A4-4 without ligand (41), but for hGST, A1-1 R9 is only ordered in the presence of a substrate (15, 42). It is thus an advantage that all of the alpha class hGSTs are amenable to this type of chemical reengineering experiment because different qualities may become useful in different types of applications. In addition, one can also potentially refine the properties of the proteins through combinatorial approaches such as gene shuffling experiments (43) due to the high homology between the isoenzymes, without losing the ability of chemical reengineering of the resulting protein scaffolds. No Apparent GS-thioester Requirements. There does not seem to be a systematic reason (size, reactivity, hydrophobicity, substitution pattern, etc.) predicting the ability of a GS-thioester to modify an alpha class GST. The reaction seems to be pretty general, and we found only one unreactive GS-thioester (GS-9). This acyl group (2,6 dimethylbenzoic acid) is di-ortho-substituted, and the likely explanation is that GS-9 is too sterically hindered to function, especially since the di-meta-substituted analogue GS-1 (3,5-dimethylbenzoic acid) is an excellent derivatization reagent for all four proteins. The size of the acyl group does not seem to be of importance since these vary from 2-thiophenecarboxylic acid (GS-13) to 5-carboxyfluorescein (GS-3). Again, this is not surprising since the natural substrates of GSTs range widely in size and shape (16). This tolerance could perhaps in part be explained by observing the crystal structures of hGST A1-1 and A4-4, where there seems to be something like a “back door” leading out from the H-site (Figure 5). The relative reactivities of the GS-thioesters correlate with the leaving group, which is GSH in all cases, and also with the pKa value of the acyl group. The reported (44) or calculated (45) pKa values of the acyl groups in the series (Supporting Information) span a range from 3.35 (2,6-dimethylbenzoic acid) to 4.80 (6-phenylhexanoic acid), but these differences are not reflected in the ability of a GS-thioester to function as an acylating reagent. The



Figure 6. The hydrophobic binding sites of hGSTs A2-2, A3-3, and A4-4 differ from that of hGST A1-1 (15) in up to nine positions, as indicated in the table. In this figure, the H-site residues are shown as CPK models with the R-carbons as balls. G-site residues are shown in stick model, again with the R-carbons as balls. The sulfur atom of the ligand S-benzylglutathione is also shown as a ball. Amino acids 101 and 131 originate from the other subunit in the dimeric protein and protrude into the G-site.

hydrophobicities of the acyl groups also differ quite substantially with log P values (the partitition coefficient between water and n-octanol) from 1.3 (4-acetamidobenzoic acid) to 3.8 (2-phenyl-4-quinolinecarboxylic acid) (Supporting Information). This trend is again not reflected in the success of modification. In our previous work we commented on the fact that there might be a need for an aromatic GS-thioester for this reaction to work (27). This was based on the observation that in our small set of three reagents, the two aromatic ones were able to acylate Y9 whereas the naturally occurring, alkyl thioester S-lactoylglutathione was not (Scheme 1 and Chart 2). In addition, rat GST A1-1 has been shown to function as a thioester hydrolase for the alkyl thioester GS-ethacrynic acid (36). To illuminate this, we included two acyl groups: phenylacetic acid (GS-16) and 6-phenylhexanoic acid (GS-17) that are closely related to benzoic acid (GSB). Even though the hydrophobicity would increase, we chose to use 6-phenylhexanoic acid instead of merely hexanoic acid since the phenyl ring is valuable as a UV marker in HPLC experiments. The reactivities of these GS-thioesters are very similar since the pKa values are 4.2, 4.3, and 4.8 for benzoic acid, phenyl acetic acid, and 6-phenylhexanoic

acid, respectively (Supporting Information). This range is included within the span of aromatic GS-thioesters that work as acylating reagents. The hydrophobicities are also within the span of the other reagents (Supporting Information) since the log P values are 1.9, 1.4, and 3.3 for benzoic acid, phenylacetic acid, and 6-phenylhexanoic acid, respectively. It does appear as if adding hGST A1-1 to GS-16 increases the rate of hydrolysis of the reagent (Table 1). However, the results suggest that even though the phenylacetic acid-derived GS-16 did not acylate hGSTs A1-1 and A4-4, the reagent with the longer spacer, GS-17, did function as an acylating reagent for all of the GSTs (Tables 1 and 2). On the basis of theses screening experiments, we can now conclude that there does not appear to be an absolute need for an aromatic GSthioester for the acylation reaction to work, something that significantly expands the versatility of the chemical reengineering approach. Besides the possible bias against di-ortho-substituted acyl groups, we cannot state any specific reagent requirements, in part because of the high success rate (>70%) in this set of GS-thioesters. Not all reagents work for all alpha class GSTs, and the reasons behind a successful modification probably depends on the combination of


protein and reagent that in turn leads to an optimal positioning of the reactive Y9 residue for the reaction to occur. Benzoic Acid-Modified hGST A1-1 Can Be Morphed into Naphthoic Acid-Modified A1-1. We found through exchange experiments that benzoic acid-modified hGST A1-1 can be altered by adding another thioester reagent (GS-2) to instead form the naphthoic acidmodified protein (Figure 4). Intriguingly, part of the premodified protein becomes deacylated during the incubation, which is odd since the benzoic acid-modified protein is completely stable during identical reaction conditions without GS-2 (27). Also, in the reaction between GS-2 and hGST A1-1, we observe naphthoic acid-modified protein but no free naphthoic acid (Table 1) as would be expected should the deacylation reaction proceed through a naphthoic acid intermediate. This finding is thus quite interesting and needs to be further studied. CONCLUSIONS

Through a combinatorial screening approach we have substantially expanded the range of GS-thioesters that are able to covalently modify the alpha class hGSTs. The set now includes fluorescent probes and a handle for chemical derivatization. The covalent modification reaction is very versatile in that a large number of GSthioesters are accepted by the alpha class GSTs, and the reaction is fast and site-directed toward one residue in the protein with minimal side reactions. This precision allows for chemical reengineering of protein scaffolds with the goal of obtaining new function through the design and synthesis of appropriate GS-thioesters. ACKNOWLEDGMENT

Kerstin S. Broo thanks the Wenner-Gren Foundation for a postdoctoral fellowship. The authors are indebted to Professor Bengt Mannervik and co-workers for contributing recombinant GSTs. This work was financially supported by The Wenner-Gren Foundation, The CarlTrygger Foundation, the Knut and Alice Wallenberg Foundation, and the Swedish Research Council. Supporting Information Available: Table 1 but with the exact figures obtained in the HPLC experiments. A table containing the experimental (44) or calculated (45) pKa and log P values of acyl groups in the panel of GSthioesters. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Bolon, D. N., Voigt, C. A., and Mayo, S. L. (2002) De novo design of biocatalysts. Curr. Opin. Chem. Biol. 6, 125-129. (2) Penning, T. M., and Jez, J. M. (2001) Enzyme redesign. Chem. Rev. 101, 3027-3046. (3) Rowan, S. J., and Sanders, J. K. (1997) Enzyme models: design and selection. Curr. Opin. Chem. Biol. 1, 483-490. (4) Hilvert, D. (2000) Critical analysis of antibody catalysis. Annu. Rev. Biochem. 69, 751-793. (5) Farinas, E. T., Bulter, T., and Arnold, F. H. (2001) Directed enzyme evolution. Curr. Opin. Biotechnol. 12, 545-551. (6) Qi, D., Tann, C. M., Haring, D., and Distefano, M. D. (2001) Generation of new enzymes via covalent modification of existing proteins. Chem. Rev. 101, 3081-3111. (7) Ren, X., Jemth, P., Board, P. G., Luo, G., Mannervik, B., Liu, J., Zhang, K., and Shen, J. (2002) A semisynthetic glutathione peroxidase with high catalytic efficiency. Selenoglutathione transferase. Chem. Biol. 9, 789-794.

Viljanen et al. (8) DeSantis, G., and Jones, J. B. (1999) Chemical modification of enzymes for enhanced functionality. Curr. Opin. Biotechnol. 10, 324-330. (9) Hamachi, I., Nagase, T., Tajiri, Y., and Shinkai, S. (1997) Incorporation of an artificial receptor into a native protein: new strategy for the design of semisynthetic enzymes with allosteric properties. Bioconjugate Chem. 8, 862-868. (10) Sheehan, D., Meade, G., Foley, V. M., and Dowd, C. A. (2001) Structure, function and evolution of glutathione transferases: implications for classification of nonmammalian members of an ancient enzyme superfamily. Biochem. J. 360, 1-16. (11) Strange, R. C., Spiteri, M. A., Ramachandran, S., and Fryer, A. A. (2001) Glutathione-S-transferase family of enzymes. Mutat. Res. 482, 21-26. (12) Armstrong, R. N. (1997) Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem. Res. Toxicol. 10, 2-18. (13) Mannervik, B. (1996) Evolution of glutathione transferases and related enzymes for the protection of cells against electrophiles. Biochem. Soc. Trans. 24, 878-880. (14) Pettigrew, N. E., and Colman, R. F. (2001) Heterodimers of glutathione S-transferase can form between isoenzyme classes pi and mu. Arch. Biochem. Biophys. 396, 225-230. (15) Sinning, I., Kleywegt, G. J., Cowan, S. W., Reinemer, P., Dirr, H. W., Huber, R., Gilliland, G. L., Armstrong, R. N., Ji, X., Board, P. G., and et al. (1993) Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes. J. Mol. Biol. 232, 192-212. (16) Hayes, J. D., and Pulford, D. J. (1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30, 445-600. (17) Lim, K., Ho, J. X., Keeling, K., Gilliland, G. L., Ji, X., Ruker, F., and Carter, D. C. (1994) Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV. Protein Sci. 3, 2233-2244. (18) Ketterer, B. (2001) A bird’s eye view of the glutathione transferase field. Chem. Biol. Interact. 138, 27-42. (19) Micaloni, C., Kong, G. K., Mazzetti, A. P., Nuccetelli, M., Antonini, G., Stella, L., McKinstry, W. J., Polekhina, G., Rossjohn, J., Federici, G., Ricci, G., Parker, M. W., and Lo Bello, M. (2003) Engineering a new C-terminal tail in the H-site of human glutathione transferase P1-1: structural and functional consequences. J. Mol. Biol. 325, 111-122. (20) Nuccetelli, M., Mazzetti, A. P., Rossjohn, J., Parker, M. W., Board, P., Caccuri, A. M., Federici, G., Ricci, G., and Lo Bello, M. (1998) Shifting substrate specificity of human glutathione transferase (from class Pi to class alpha) by a single point mutation. Biochem. Biophys. Res. Commun. 252, 184-189. (21) Pettersson, P. L., Johansson, A. S., and Mannervik, B. (2002) Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase. J. Biol. Chem. 277, 30019-30022. (22) Widersten, M., Hansson, L. O., Tronstad, L., and Mannervik, B. (2000) Use of phage display and transition-state analogues to select enzyme variants with altered catalytic properties: glutathione transferase as an example. Methods Enzymol. 328, 389-404. (23) Nilsson, L. O., Gustafsson, A., and Mannervik, B. (2000) Redesign of substrate-selectivity determining modules of glutathione transferase A1-1 installs high catalytic efficiency with toxic alkenal products of lipid peroxidation. Proc. Natl. Acad. Sci. U.S.A. 97, 9408-9412. (24) Hansson, L. O., and Mannervik, B. (2000) Use of chimeras generated by DNA shuffling: probing structure-function relationships among glutathione transferases. Methods Enzymol. 328, 463-477. (25) Hansson, L. O., Widersten, M., and Mannervik, B. (1999) An approach to optimizing the active site in a glutathione transferase by evolution in vitro. Biochem. J. 344 Pt 1, 93100. (26) Mannervik, B., Cameron, A. D., Fernandez, E., Gustafsson, A., Hansson, L. O., Jemth, P., Jiang, F., Jones, T. A., Larsson,

Expanding the Range of GST Acylation Reagents A. K., Nilsson, L. O., Olin, B., Pettersson, P. L., Ridderstrom, M., Stenberg, G., and Widersten, M. (1998) An evolutionary approach to the design of glutathione-linked enzymes. Chem. Biol. Interact. 111-112, 15-21. (27) Ha˚kansson, S., Viljanen, J., Broo, K. S. (2003) Programmed delivery of novel functional groups to the alpha class glutathione transferases. Biochemistry 42, 10260-10268. (28) Davies, R. R., and Distefano, M. D. (1997) A semisynthetic metalloenzyme based on a protein cavity that catalyzes the enantioselective hydrolysis of ester and amide substrates. J. Am. Chem. Soc. 119, 11643-11652. (29) Haring, D., and Distefano, M. D. (2001) Enzymes by design: chemogenetic assembly of transamination active sites containing lysine residues for covalent catalysis. Bioconjug. Chem. 12, 385-390. (30) Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595-598. (31) Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77. (32) Habig, W. H., and Jakoby, W. B. (1981) Assays for differentiation of glutathione S-transferases. Methods Enzymol. 77, 398-405. (33) Gustafsson, A., and Mannervik, B. (1999) Benzoic acid derivatives induce recovery of catalytic activity in the partially inactive Met208Lys mutant of human glutathione transferase A1-1. J. Mol. Biol. 288, 787-800. (34) Pettersson, P. L., and Mannervik, B. (2001) The role of glutathione in the isomerization of delta 5-androstene-3, 17dione catalyzed by human glutathione transferase A1-1. J. Biol. Chem. 276, 11698-704. (35) Johansson, A. S., and Mannervik, B. (2001) Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones. J. Biol. Chem. 276, 33061-5. (36) Dietze, E. C., Grillo, M. P., Kalhorn, T., Nieslanik, B. S., Jochheim, C. M., and Atkins, W. M. (1998) Thiol ester

Bioconjugate Chem., Vol. 15, No. 4, 2004 727 hydrolysis catalyzed by glutathione S-transferase A1-1. Biochemistry 37, 14948-57. (37) Johansson, A. S., and Mannervik, B. (2002) Active-site Residues Governing High Steroid Isomerase Activity in Human Glutathione Transferase A3-3. J. Biol. Chem. 277, 16648-54. (38) Dreij, K., Sundberg, K., Johansson, A. S., Nordling, E., Seidel, A., Persson, B., Mannervik, B., and Jernstrom, B. (2002) Catalytic activities of human alpha class glutathione transferases toward carcinogenic dibenzo[a, l]pyrene diol epoxides. Chem. Res. Toxicol. 15, 825-31. (39) Hubatsch, I., Ridderstrom, M., and Mannervik, B. (1998) Human glutathione transferase A4-4: an alpha class enzyme with high catalytic efficiency in the conjugation of 4-hydroxynonenal and other genotoxic products of lipid peroxidation. Biochem. J. 330 (Pt 1), 175-9. (40) Wang, J., Bauman, S., and Colman, R. F. (2000) Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4(succinimidyl)benzophenone]. J. Biol. Chem. 275, 5493-503. (41) Bruns, C. M., Hubatsch, I., Ridderstrom, M., Mannervik, B., and Tainer, J. A. (1999) Human glutathione transferase A4-4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products. J. Mol. Biol. 288, 427-39. (42) Cameron, A. D., Sinning, I., L’Hermite, G., Olin, B., Board, P. G., Mannervik, B., and Jones, T. A. (1995) Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate. Structure 3, 717-27. (43) Kolkman, J. A., and Stemmer, W. P. (2001) Directed evolution of proteins by exon shuffling. Nat. Biotechnol. 19, 423-8. (44) http://chemfinder.cambridgesoft.com/result.asp. (45) CompuDrug, I., Inc. (1995), 705, Grandview Drive, South San Francisco, CA 94080.

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Combinatorial Chemical Reengineering of the Alpha Class

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