Human Glutathione Transferase A1-1 Research Articles

A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1.

Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs.

The Interaction of Human Glutathione Transferase GSTA1-1 with Reactive Dyes

Human glutathione transferase A1-1 (hGSTA1-1) contributes to developing resistance to anticancer drugs and, therefore, is promising in terms of drug-design targets for coping with this phenomenon. In the present study, the interaction of anthraquinone and diazo dichlorotriazine dyes (DCTD) with hGSTA1-1 was investigated. The anthraquinone dye Procion blue MX-R (PBMX-R) appeared to interact with higher affinity and was selected for further study. The enzyme was specifically and irreversibly inactivated by PBMX-R, following a biphasic pseudo-first-order saturation kinetics, with approximately 1 mol of inhibitor per mol of the dimeric enzyme being incorporated. Molecular modeling and protein chemistry data suggested that the modified residue is the Cys112, which is located at the entrance of the solvent channel at the subunits interface. The results suggest that negative cooperativity exists upon PBMX-R binding, indicating a structural communication between the two subunits. Kinetic inhibition analysis showed that the dye is a competitive inhibitor towards glutathione (GSH) and mixed-type inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). The present study results suggest that PBMX-R is a useful probe suitable for assessing by kinetic means the drugability of the enzyme in future drug-design efforts.

The Interaction of the Flavonoid Fisetin with Human Glutathione Transferase A1-1.

Glutathione transferases (GSTs) are a family of Phase II detoxification enzymes that are involved in the development of the multidrug resistance (MDR) mechanism in cancer cells and therefore affect the clinical outcome of cancer chemotherapy. The discovery of nontoxic natural compounds as inhibitors for GSTs is a promising approach for chemosensitizing and reversing MDR. Fisetin (7,3′,4′-flavon-3-ol) is a plant flavonol present in many plants and fruits. In the present work, the interaction of fisetin with human glutathione transferase A1-1 (hGSTA1-1) was investigated. Kinetic analysis revealed that fisetin is a reversible inhibitor for hGSTA1-1 with IC50 1.2 ± 0.1 μΜ. It functions as a mixed-type inhibitor toward glutathione (GSH) and as a noncompetitive inhibitor toward the electrophile substrate 1-chloro-2,4-dinitrobenzene (CDNB). In silico molecular modeling and docking predicted that fisetin binds at a distinct location, in the solvent channel of the enzyme, and occupies the entrance of the substrate-binding sites. Treatment of proliferating human epithelial colorectal adenocarcinoma cells (CaCo-2) with fisetin causes a reduction in the expression of hGSTA1-1 at the mRNA and protein levels. In addition, fisetin inhibits GST activity in CaCo-2 cell crude extract with an IC50 (2.5 ± 0.1 μΜ), comparable to that measured using purified recombinant hGSTA1-1. These actions of fisetin can provide a synergistic role toward the suppression and chemosensitization of cancer cells. The results of the present study provide insights into the development of safe and effective GST-targeted cancer chemosensitizers.

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site-Specific Elucidation of Hidden Dynamics in Enzymes.

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-CuII NTA label to obtain site-specific information on dynamics for both an α-helix and β-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure site-specific dynamics using room temperature lineshape analysis.

A Microplate-based Platform with Immobilized Human Glutathione Transferase A1-1 for High-throughput Screening of Plant-origin Inhibitors.

Glutathione transferases (GSTs) catalyze the conjugation of glutathione (GSH) to endogenous and xenobiotic electrophilic compounds and have been involved in the development of resistance toward cancer chemotherapeutic drugs and in the etiology, pathology and progression of several other diseases. In the present work, the human isoenzyme GSTA1-1 (hGSTA1-1) was used to assemble a microplate-based platform for high-throughput screening of natural productbased inhibitors from plant extracts. The enzyme was immobilized using sol-gel chemistry and deposited as a layer at the bottom surface of 96-well format ELISA microplate. The sensing signal was based on the inhibition of the colorimetric reaction between 1-chloro-dinitrobenzene (CDNB) and GSH, catalyzed by the sol-gel entrapped enzyme. As a proof of concept, the system was used for screening aqueous extracts from medicinal and aromatic plants with excellent reproducibility (approximately 95%). The operational simplicity and accuracy of this system, suggest that it can be explored as a bioanalytical tool with potential use in drug design and development efforts for finding new sources of GST inhibitors useful in chemomodulation of cancer drugs.

Abstract 5238: Characterization of the enzyme generating the cholesterol metabolite and tumor suppressor dendrogenin A in the breast and its deregulations in breast cancer

Abstract Dendrogenin A (DDA) is a tumor suppressor metabolite identified in human tissues that arises from the conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA) by a yet unidentified enzyme. DDA is present in the normal breast but its levels were found drastically decreased in breast tumors, showing that a deregulation of DDA metabolism occurred during breast carcinogenesis. It was shown that DDA displayed chemopreventive and anticancer properties (de Medina et al, Nat Commun, 2013; Voisin et al, PNAS, 2017; Segala et al, Nat Commun, in press). In addition, DDA blocks the biosynthesis of a newly identified cholesterol tumor promoter named 6-oxo-cholestan-3β,5α-diol (OCDO) (Voisin et al, PNAS, 2017). DDA and OCDO arise from 5,6-EC. We showed the existence of a metabolic balance between these two 5,6-EC derivatives in normal breast and BC that controls or stimulates BC progression (Silvente-Poirot & Poirot, Science, 2014, Voisin et al, PNAS, 2017). We addressed here the question of the identification and characterization of the DDA synthase (DDAS) and we determined whether its expression could reflect DDA levels in patient breast tumor and normal tissue. We report that the recombinant human glutathione transferase A1-1 (GST A1-1) produced DDA from 5,6α-EC and histamine (HA). The chemical characterization of the DDA product was performed by chromatography and mass spectrometry fragmentation. DDAS activity was found to be a new and important activity of GST A1-1 in addition to known glutathione transferase and steroid isomerase activities. The measured Michaelis constants of GST A1-1 for its new substrates were: Km5,6α-EC=0.27±0.05 µM and KmHA=0.35±0.3 µM, and the maximum velocity for the transformation of each substrates Vm5,6α-EC=0.81±0.2 µmol.min-1.mg and VmHA=0.66±0.2 µmol.min-1.mg. Interestingly, we showed that OCDO and other ring-B oxysterols, as well as several natural substrates and product of the GST A1-1, were potent inhibitors of DDAS activity while xenobiotics substrates of GST, and side chain oxysterols were not. Patient BC samples (n=50) showed significant decreased DDA levels and lower GST A1-1 protein expression compared to normal matched tissues, indicating that the decreased production of DDA in tumors is due to decreased expression of its enzyme. The analyses of two human BC mRNA databases from the Barts Cancer Institute (London, UK) and the Curie Institute (Paris, France) showed that the expression of GST A1-1 was lost in ER(+) BC tumors compared to normal breast tissue. Interestingly, DDAS was selectively expressed in the cytoplasm of epithelial cells from lactating ducts and lobular terminal units. Since these cells are the origin of most BC, the loss of DDAS expression and DDA biosynthesis combine to OCDO production, which controls DDAS activity, may constitute a major oncogenic process leading to BC development in human. Citation Format: Marc Poirot, Emmanuel Noguer, Florence Dalenc, Regis Soules, Lisa Barrett, Arnaud Rives, Hye-Young Kim, Brigitta Sjödin, Camille Franchet, Pilippe Rochaix, Raphaelle Duprez-Paumier, Magali Lacroix-Triki, Thomas Filleron, Leonor Chaltiel, Louise Jones, Emanuala Gadaleta, Claude Chalala, Sergio Roman-Roman, Thierry Dubois, Ned A. Porter, Bengt Mannervik, Michel Record, Sandrine Silvente-Poirot. Characterization of the enzyme generating the cholesterol metabolite and tumor suppressor dendrogenin A in the breast and its deregulations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5238.

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability

BackgroundGlutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. MethodsA library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). ResultsActivity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher kcat/Km and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Å and 2.3Å resolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. ConclusionsDNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. General significanceSuch an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors.

Synthesis and Study of 2-(Pyrrolesulfonylmethyl)-N-arylimines: A New Class of Inhibitors for Human Glutathione Transferase A1-1

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (K(i(9)) = 71 ± 4 μM) at the primary site competitively vs CDNB. Derivative 4 binds (K(i(4)) = 135 ± 27 μM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure.

Energetics of ligand binding to human glutathione transferase A1-1: Tyr-9 associated localisation of the C-terminal helix is ligand-dependent

A C-terminal helix (α9) adjacent to the active site on each subunit is a structural feature unique to the alpha isoform of glutathione transferases which contributes to the catalytic and ligandin functions of the enzyme. The ionisation state of Tyr-9, a residue critical to catalysis, influences α9 dynamics, although the mechanism is poorly understood. In this study, isothermal titration calorimetry was used to probe the binding energetics of G-site (glutathione and glutathione sulfonate) and H-site (ethacrynic acid) ligands to wild-type and a Y9F mutant of human glutathione transferase A1-1. Although previous studies have reported a favourable entropic component to the binding of conjugates occupying both sites, our data reveal that ligand binding is enthalpically driven when either the G- or H-site is occupied independently. Also, heat capacity changes demonstrate that α9 is fully localised by H-site but not G-site occupation. The Tyr-9 hydroxyl group contributes significantly to ligand binding energetics, although the effect differs between the two binding sites. G-site binding is made slightly enthalpically more favourable and entropically less favourable by the Y9F mutation. Binding to the H-site is more dramatically affected, with the K d for ethacrynic acid increasing 5 fold despite a more favourable Δ S. The heat capacity change is more negative for G-site binding in the absence of the Tyr-9 hydroxyl (ΔΔ C p = − 0.73 kJ mol −1 K −1), but less negative for H-site binding to the Y9F mutant (ΔΔ C p = 0.63 kJ mol −1 K −1). This suggests that the relationship between Tyr-9 and α9 is not independent of the ligand. Rather, Tyr-9 appears to function in orienting the ligand optimally for α9 closure.

Arginine 15 stabilizes an S NAr reaction transition state and the binding of anionic ligands at the active site of human glutathione transferase A1-1

Arg15, conserved in class Alpha GSTs (glutathione transferases), is located at the interface between the G- and H-sites of the active site where its cationic guanidinium group might play a role in catalysis and ligand binding. Arg15 in human GSTA1-1 was replaced with a leucine and crystallographic, spectroscopic, thermodynamic and molecular docking methods were used to investigate the contribution made by Arg15 towards (i) the binding of glutathione (GSH) to the G-site, (ii) the p K a of the thiol group of GSH, (iii) the stabilization of an analog of the anionic transition state of the S NAr reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, and, (iv) the binding of the anionic non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to the H-site. While the R15L mutation substantially diminishes the CDNB–GSH conjugating activity of the enzyme, it has little effect on protein structure and stability. Arg15 does not contribute significantly towards the enzyme's affinity for GSH but does determine the reactivity of GSH by reducing the thiol's p K a from 7.6 to 6.6. The anionic σ-complex formed between GSH and 1,3,5-trinitrobenzene is stabilized by Arg15, suggesting that it also stabilizes the transition state formed in the S NAr reaction between GSH and CDNB. The trinitrocyclohexadienate moiety of the σ-complex binds the H-site where the catalytic residue, Tyr9, was identified to hydrogen bond to an o-nitro group of the σ-complex. The affinity for ANS at the H-site is decreased about 3-fold by the R15L mutation implicating the positive electrostatic potential of Arg15 in securing the organic anion at this site.

Structural Analysis of a Glutathione Transferase A1-1 Mutant Tailored for High Catalytic Efficiency with Toxic Alkenals

The specificity of human glutathione transferase (GST) A1-1 is drastically altered to favor alkenal substrates in the GIMFhelix mutant designed to mimic first-sphere interactions utilized by GSTA4-4. This redesign serves as a model for improving our understanding of the structural determinants that contribute to the distinct specificities of alpha class GSTs. Herein we report the first crystal structures of GIMFhelix, both in complex with GSH and in apo form at 1.98 and 2.38 A resolution. In contrast to the preorganized hydrophobic binding pocket that accommodates alkenals in GSTA4-4, GSTA1-1 includes a dynamic alpha9 helix that undergoes a ligand-dependent localization to complete the active site. Comparisons of the GIMFhelix structures with previously reported structures show a striking similarity with the GSTA4-4 active site obtained within an essentially GSTA1-1 scaffold and reveal the alpha9 helix assumes a similar localized structure regardless of active site occupancy in a manner resembling that of GSTA4-4. However, we cannot fully account for all the structural elements important in GSTA4-4 within the mutant's active site. The contribution of Phe10 to the Tyr212-Phe10-Phe220 network prevents complete C-terminal closure and demonstrates that the presence of Phe10 within the context of a GSTA4-4-like active site may ultimately hinder Phe220, a key C-terminal residue, from effectively contributing to the active site. In total, these results illustrate the remaining structural differences presumably reflected in the previously reported catalytic efficiencies of GIMFhelix and GSTA4-4 and emphasize the F10P mutation as being necessary to completely accomplish the transformation to a highly specific GST from the more promiscuous GSTA1-1 enzyme.

Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates

Efforts are being made to engineer enzymes with enhanced activities against haloalkanes, a toxicologically important class of compounds widely used and frequently occurring in the environment. Here we describe a facile, inexpensive, and robust method for the screening of libraries of mutated enzymes with iodoalkane substrates. Iodide formed in the enzymatic reaction is oxidized to iodine, which in the presence of starch gives blue color that can be measured at 610nm or scored with the human eye. The assay can be performed with enzymes in crude cell lysates in 96-wells microtiter plates. Expression clones of several glutathione transferases showed diverse activities with different iodoalkanes, and a mutant library of human glutathione transferase A1-1 expressed variants with enhanced substrate selectivities.

Surface-Assisted Delivery of Fluorescent Groups to hGST A1-1 and a Lysine Mutant

Human glutathione transferase (hGST) A1-1 and a lysine mutant (A216K) can both be rapidly and site-specifically acylated on Y9 and K216, respectively, using a range of thiolesters of glutathione (GS-thiolesters) as modifying reagents. The present investigation was aimed at developing a method with which to deliver a fluorescent acyl group from a solid support under conditions compatible with standard protein purification schemes. A number of fluorescent GS-thiolesters with modified peptide backbones were therefore prepared and tested for reactivity toward hGST A1-1 and the A216K mutant. Substitutions at the alpha-NH2 part of the glutathione backbone were not tolerated by the proteins. However, two fluorescent reagents that carry a biotin moiety at the C-terminal part of glutathione were found through MALDI-MS experiments to react in solution with Y9 of the wild-type protein and one reagent with K216 of A216K. The reaction can take place in the presence of glutathione and even in a crude E. coli lysate of cells expressing A216K. Delivery of the fluorescent group to Y9 or K216 was possible using NeutrAvidin (NA) beads that had been preincubated with biotinylated reagent. Alternatively, excess reagent can be removed by a brief incubation with NA beads. We have thus now developed a system for protein labeling with easy removal of excess and used up low-molecular weight reagent. This strategy can conceivably be utilized in future protein purification and labeling experiments.

New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix

Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.

A promiscuous glutathione transferase transformed into a selective thiolester hydrolase

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.

The intersubunit lock-and-key motif in human glutathione transferase A1-1: role of the key residues Met51 and Phe52 in function and dimer stability

The dimeric structure of certain cytosolic GSTs (glutathione S-transferases) is stabilized by a hydrophobic lock-and-key motif at their subunit interface. In hGSTA1-1 (human class Alpha GST with two type-1 subunits), the key consists of two residues, Met51 and Phe52, that fit into a hydrophobic cavity (lock) in the adjacent subunit. SEC (size-exclusion chromatography)-HPLC, far-UV CD and tryptophan fluorescence of the M51A and M51A/F52S mutants indicated the non-disruptive nature of these mutations on the global structure. While the M51A mutant retained 80% of wild-type activity, the activity of the M51A/F52S was markedly diminished, indicating the importance of Phe52 in maintaining the correct conformation at the active site. The M51A and M51A/F52S mutations altered the binding of ANS (8-anilinonaphthalene-l-sulphonic acid) at the H-site by destabilizing helix 9 in the C-terminal region. Data from urea unfolding studies show that the dimer is destabilized by both mutations and that the dimer dissociates to aggregation-prone monomers at low urea concentrations before global unfolding. Although not essential for the assembly of the dimeric structure of hGSTA1-1, both Met51 and Phe52 in the intersubunit lock-and-key motif play important structural roles in maintaining the catalytic and ligandin functions and stability of the GST dimer.

Nucleophile selectivity in the acyl transfer reaction of a designed enzyme

The acyl transfer reaction of S-glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1-1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme-catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar pKa values depended on the structure of the aliphatic chain, and 1-propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on pKa as 2,2,2-trifluoroethanol, with a pKa of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1-propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities.

Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants

Previously, we discovered that human glutathione transferase (hGST) A1-1 could be site-specifically acylated on a tyrosine residue (Y9) to form ester products using thiolesters of glutathione (GS-thiolesters) as acylating reagents. Out of a total of 20 GS-thiolester reagents tested, 15 (75%) are accepted by hGST A1-1 and thus this is a very versatile reaction. The present investigation was aimed at obtaining a more stable product, an amide bond, between the acyl group and the protein, in order to further increase the value of the reaction. Three lysine mutants (Y9K, A216K, and Y9F/A216K) were therefore prepared and screened against a panel of 18 GS-thiolesters. The Y9K mutant did not react with any of the reagents. The double mutant Y9F/A216K reacted with only one reagent, but in contrast, the A216K mutant could be acylated at the introduced lysine 216 with eight (44%) of the GS-thiolesters. The reaction can take place in the presence of glutathione and even in a crude cell lysate for five (28%) of the reagents. Through the screening process we obtained some basic rules relating to reagent requirements. We have thus produced a mutant (A216K) that can be rapidly and site-specifically modified at a lysine residue to form a stable amide linkage with a range of acyl groups. One of the successful reagents is a fluorophore that potentially can be used in downstream protein purification and protein fusion applications.

Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1.

A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

Glutathione induces helical formation in the carboxy terminus of human glutathione transferase A1-1.

The structure and dynamic properties of the C-terminal region of the human class alpha glutathione transferase A1-1 have been investigated with high-resolution NMR methods. On the basis of crystallographic and fluorescence measurements, this 13-residue segment of the enzyme is presumed to be disordered in the unliganded enzyme. When the product or product analogue is bound, a C-terminal alpha-helix is observed in crystal structures. Conflicting data exists regarding the structure of this region when one of the substrates, glutathione (GSH), is bound. The NMR studies presented here show that in the unliganded protein, this region of the protein samples different conformations, most likely an ensemble of helix-like structures. Addition of either GSH or the conjugate between GSH and ethacrynic acid (EASG) causes this segment to become a stable alpha-helix. In the GSH complex, the ends of this helix exhibit dynamic behavior on both the millisecond and nanosecond time scales. In contrast, there is no evidence of millisecond dynamics in the EASG complex. The ligand-induced ordering of the enzyme reduces the intrinsic affinity of the enzyme for its product, facilitating enzymatic turnover.