Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

The present study characterized conserved residues in a GST (glutathione transferase) in the active-site region that interacts with glutathione. This region of the active site is near the glycine moiety of glutathione and consists of a hydrogen bond network. In the GSTD (Delta class GST) studied, adGSTD4-4, the network consisted of His(38), Met(39), Asn(47), Gln(49), His(50) and Cys(51). In addition to contributing to glutathione binding, this region also had major effects on enzyme catalysis, as shown by changes in kinetic parameters and substrate-specific activity. The results also suggest that the electron distribution of this network plays a role in stabilization of the ionized thiol of glutathione as well as impacting on the catalytic rate-limiting step. This area constitutes a second glutathione active-site network involved in glutathione ionization distinct from a network previously observed interacting with the glutamyl end of glutathione. This second network also appears to be functionally conserved in GSTs. In the present study, His(50) is the key basic residue stabilized by this network, as shown by up to a 300-fold decrease in k(cat) and 5200-fold decrease in k(cat)/K(m) for glutathione. Although these network residues have a minor role in structural integrity, the replaced residues induced changes in active-site topography as well as generating positive co-operativity towards glutathione. Moreover, this network at the glycine moiety of GSH (glutathione) also contributed to the 'base-assisted deprotonation model' for GSH ionization. Taken together, the results indicate a critical role for the functionally conserved basic residue His(50) and this hydrogen bond network in the active site.     

Bioinformatic analysis and in vitro site-directed mutagenesis of conserved amino acids in BphK, a specific bacterial glutathione transferase

Glutathione transferases [GSTs: EC 2.5.1.18)] are ubiquitous multifunctional prokaryotic and eukaryotic enzymes involved in the cellular detoxification and excretion of a large variety of compounds. However, our understanding of the role of bacterial GSTs in metabolism is still in its infancy. The association of bacterial GST DNA with other genes involved in degradation of toxic pollutants, including polychlorinated biphenyls (PCBs), indirectly suggests a role for bacterial GSTs in biodegradation. Previously, in this laboratory, a specific bacterial GST, BphK^LB400 isolated from Burkholderia xenovorans LB400, was shown to be capable of dehalogenating chlorinated organic substrates rendering them less toxic. However, little is known about the specific amino acids in BphK^LB400 involved in catalysis in vitro. In this study, bioinformatic analysis of BphK^LB400 and other bacterial GSTs, including PCB degraders, identified a number of amino acids that were identical in all bacterial GST sequences analysed. Two amino acids, Cys10 and His106, were selected for in vitro site-directed mutagenesis studies. In vitro GST activity assay results suggest that these two amino acids play a role in determining the catalytic activity of BphK^LB400. Studies of bacterial cell extracts expressing BphK^LB400 (wildtype and mutant) identified a specific mutant, Cys10Phe, with increased GST activity towards 1-chloro-2,4-dinitrobenzene (the model substrate for GSTs). BphK^LB400 (mutant) with increased activity towards toxic chlorinated organic compounds could have potential for bioremediation of contaminated soil in the environment.     

Knowledge-based modeling of a bacterial dichloromethane dehalogenase

A three-dimensional structural model of the dichloromethane dehalogenase (DCMD) from Methylophilus sp. DM11 is constructed based on sequence similarities to the glutathione S-transferases (GSTs). To maximize sequence identity and minimize gaps in the alignment, a hybrid approach is used that takes advantage of the increased homology found between DM11 and domain I of the sheep blowfly θ class GST (residues 1–79) and domain II of the human α class GST (residues 81–222). The resulting structure has Cα root mean square deviations of 1.16 Å in domain I and 1.83 Å in domain II from the template GSTs, which compare well to those seen in other GST interclass comparisons. The model is further applied to explore the structural basis for substrate binding and catalysis. A conserved network of hydrogen bonds is described that binds glutathione to the G site, placing the thiol group in a suitable location for nucleophilic attack of dichloromethane. A mechanism is proposed that involves activation through a hydrogen bond interaction between Ser12 and glutathione, similar to that found in the θ-GSTs. The model also demonstrates how aromatic residues in the hydrophobic site (H site) could play a role in promoting catalysis: His116 and Trp117 are ideally situated to accept a growing negative charge on a chlorine of dichloromethane, stabilizing displacement. This scheme is consistent with experimental results of single-point mutations and comparisons with other GST structures and mechanisms. Proteins 28:217–226, 1997. © 1997 Wiley-Liss Inc.     

Binding and kinetic mechanisms of the Zeta class glutathione transferase

The Zeta class of glutathione transferases (GSTs) has only recently been discovered and hence has been poorly characterized. Here we investigate the substrate binding and kinetic mechanisms of the human Zeta class GSTZ1c-1c by means of pre-steady state and steady-state experiments and site-directed mutagenesis. Binding of GSH occurs at a very low rate compared with that observed for the more recently evolved GSTs (Alpha, Mu, and Pi classes). Moreover, the single step binding mechanism observed in this enzyme is reminiscent of that found for the Theta class enzyme, whereas the Alpha, Mu, and Pi classes have adopted a multistep binding mechanism. Replacement of Cys16 with Ala increases the rate of GSH release from the active site causing a 10-fold decrease of affinity toward GSH. Cys16 also plays a crucial role in co-substrate binding; the mutant enzyme is unable to bind the carcinogenic substrate dichloroacetic acid in the absence of GSH. However, both substrate binding and GSH activation are not rate-limiting in catalysis. A peculiarity of the hGSTZ1c-1c is the half-site activation of bound GSH. This suggests a primitive monomer-monomer interaction that, in the recently diverged GSTP1-1, gives rise to a sophisticated cooperative mechanism that preserves the catalytic efficiency of this GST under stress conditions.     

An electron-sharing network involved in the catalytic mechanism is functionally conserved in different glutathione transferase classes

In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl alpha-carboxylate group of glutathione, the positively charged Arg-66, and the negatively charged Asp-100. This ionic interaction is stabilized by a network of hydrogen bonds from Ser-65, Thr-158, Thr-162, and a conserved water-mediated contact. This alternating ionic bridge interaction between negatively and positively charged residues stabilized by a network of hydrogen bonding we have named an electron-sharing network. We show that the electron-sharing network assists the glutamyl alpha-carboxylate of glutathione to function as a catalytic base accepting the proton from the thiol group forming an anionic glutathione, which is a crucial step in the glutathione transferase (GST) catalysis. Kinetic studies demonstrate that the mutation of electron-sharing network residues results in a decreased ability to lower the pKa of the thiol group of glutathione. Although the residues that contribute to the electron-sharing network are not conserved in the primary sequence, structural characterizations indicate that the presence of the network can be mapped to the same region in all GST classes. A structural diversification but functional conservation suggests a significant role for the electron-sharing network in catalysis as the purpose was maintained during the divergent evolution of GSTs. This network appears to be a functionally conserved motif that contributes to the "base-assisted deprotonation" model suggested to be essential for the glutathione ionization step of the catalytic mechanism.     

Structural and mechanistic insights into unusual thiol disulfide oxidoreductase

Cytoplasmic desulfothioredoxin (Dtrx) from the anaerobe Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thiol disulfide oxidoreductase family. The active site of Dtrx contains a particular consensus sequence, CPHC, never seen in the cytoplasmic thioredoxins and generally found in periplasmic oxidases. Unlike canonical thioredoxins (Trx), Dtrx does not present any disulfide reductase activity, but it presents instead an unusual disulfide isomerase activity. We have used NMR spectroscopy to gain insights into the structure and the catalytic mechanism of this unusual Dtrx. The redox potential of Dtrx (-181 mV) is significantly less reducing than that of canonical Trx. A pH dependence study allowed the determination of the pK(a) of all protonable residues, including the cysteine and histidine residues. Thus, the pK(a) values for the thiol group of Cys(31) and Cys(34) are 4.8 and 11.3, respectively. The His(33) pK(a) value, experimentally determined for the first time, differs notably as a function of the redox states, 7.2 for the reduced state and 4.6 for the oxidized state. These data suggest an important role for His(33) in the molecular mechanism of Dtrx catalysis that is confirmed by the properties of mutant DtrxH33G protein. The NMR structure of Dtrx shows a different charge repartition compared with canonical Trx. The results presented are likely indicative of the involvement of this protein in the catalysis of substrates specific of the anaerobe cytoplasm of DvH. The study of Dtrx is an important step toward revealing the molecular details of the thiol-disulfide oxidoreductase catalytic mechanism.     

Functional divergence in the glutathione transferase superfamily in plants. Identification of two classes with putative functions in redox homeostasis in Arabidopsis thaliana

Searches with the human Omega glutathione transferase (GST) identified two outlying groups of the GST superfamily in Arabidopsis thaliana which differed from all other plant GSTs by containing a cysteine in place of a serine at the active site. One group consisted of four genes, three of which encoded active glutathione-dependent dehydroascorbate reductases (DHARs). Two DHARs were predicted to be cytosolic, whereas the other contained a chloroplast targeting peptide. The DHARs were also active as thiol transferases but had no glutathione conjugating activity. Unlike most other GSTs, DHARs were monomeric. The other class of GST comprised two genes termed the Lambda GSTs (GSTLs). The recombinant GSTLs were also monomeric and had glutathione-dependent thiol transferase activity. One GSTL was cytosolic, whereas the other was chloroplast-targeted. When incubated with oxidized glutathione, the putative active site cysteine of the GSTLs and cytosolic DHARs formed mixed disulfides with glutathione, whereas the plastidic DHAR formed an intramolecular disulfide. DHAR S-glutathionylation was consistent with a proposed catalytic mechanism for dehydroascorbate reduction. Roles for the cytosolic DHARs and GSTLs as antioxidant enzymes were also inferred from the induction of the respective genes following exposure to chemicals and oxidative stress.     

Non-essentiality of cysteine and histidine residues for the activity of human class PI glutathione S-transferase.

In order to examine the roles of cysteine and histidine residues in the activity of human class Pi glutathione S-transferase (GST pi), site-directed mutagenesis was used to replace each of the four cysteine residues (at positions 14, 47, 101 and 169) with serine and each of the two histidine residues (at positions 71 and 162) with asparagine using a cDNA for the enzyme (Kano, T. et al. (1987) Cancer Res., 47, 5626-5630) and an E. coli expression system. The replacements of Cys101, Cys169, His71 and His162 did not affect the GSH-conjugating activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid. On the other hand, the activities were partly decreased by the replacements of Cys47 and Cys14. These results indicated that the cysteine and histidine residues in GST pi are not essential for the catalytic activity, although Cys47 and Cys14 may contribute to some extent to the catalytic efficiency.     

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse,ancient protein super family using motif search and structural modeling

Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor 1γ (EF1γ) contains an N-terminal domain related to class θ glutathione S-transferases (GST). GST-like proteins related to class θ comprise a large group including, in addition to typical GSTs and EF1γ, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and β-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (α, μ, and π). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EF1γ is likely to form a fold very similar to that in the known structures of class α, μ, and π GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GST-like domain in EF1γ is enzymatically active and that to exhibit GST activity, EF1γ has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EF1 and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.     

Understanding mammalian glutathione peroxidase 7 in the light of its homologs

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (C_P). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the C_P is formed with an alleged resolving Cys (C_R) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional C_R suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which C_P both reduces H₂O₂ and oxidizes PDI, and that the C_P-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.     

Glutamate-64, a newly identified residue of the functionally conserved electron-sharing network contributes to catalysis and structural integrity of glutathione transferases

In Anopheles dirus glutathione transferase D3-3, position 64 is occupied by a functionally conserved glutamate residue, which interacts directly with the gamma-glutamate moiety of GSH (glutathione) as part of an electron-sharing network present in all soluble GSTs (glutathione transferases). Primary sequence alignment of all GST classes suggests that Glu64 is one of a few residues that is functionally conserved in the GST superfamily. Available crystal structures as well as consideration of the property of the equivalent residue at position 64, acidic or polar, suggest that the GST electron-sharing motif can be divided into two types. Electrostatic interaction between the GSH glutamyl and carboxylic Glu64, as well as with Arg66 and Asp100, was observed to extend the electron-sharing motif identified previously. Glu64 contributes to the catalytic function of this motif and the 'base-assisted deprotonation' that are essential for GSH ionization during catalysis. Moreover, this residue also appears to affect multiple steps in the enzyme catalytic strategy, including binding of GSH, nucleophilic attack by thiolate at the electrophilic centre and product formation, probably through active-site packing effects. Replacement with non-functionally-conserved amino acids alters initial packing or folding by favouring aggregation during heterologous expression. Thermodynamic and reactivation in vitro analysis indicated that Glu64 also contributes to the initial folding pathway and overall structural stability. Therefore Glu64 also appears to impact upon catalysis through roles in both initial folding and structural maintenance.     

Insights into the catalytic mechanism of glutathione S-transferase: the lesson from Schistosoma haematobium

Glutathione S-transferases (GSTs) are involved in detoxification of xenobiotic compounds and in the biosynthesis of important metabolites. All GSTs activate glutathione (GSH) to GS(-); in many GSTs, this is accomplished by a Tyr at H-bonding distance from the sulfur of GSH. The high-resolution structure of GST from Schistosoma haematobium revealed that the catalytic Tyr occupies two alternative positions, one external, involving a pi-cation interaction with the conserved Arg21, and the other inside the GSH binding site. The interaction with Arg21 lowers the pK(a) of the catalytic Tyr10, as required for catalysis. Examination of several other GST structures revealed the presence of an external pocket that may accommodate the catalytic Tyr, and suggested that the change in conformation and acidic properties of the catalytic Tyr may be shared by other GSTs. Arginine and two other residues of the external pocket constitute a conserved structural motif, clearly identified by sequence comparison.     

Disulfide bond structure and domain organization of yeast beta(1,3)-glucanosyltransferases involved in cell wall biogenesis

The Gel/Gas/Phr family of fungal beta(1,3)-glucanosyltransferases plays an important role in cell wall biogenesis by processing the main component beta(1,3)-glucan. Two subfamilies are distinguished depending on the presence or absence of a C-terminal cysteine-rich domain, denoted "Cys-box." The N-terminal domain (NtD) contains the catalytic residues for transglycosidase activity and is separated from the Cys-box by a linker region. To obtain a better understanding of the structure and function of the Cys-box-containing subfamily, we identified the disulfide bonds in Gas2p from Saccharomyces cerevisiae by an improved mass spectrometric methodology. We mapped two separate intra-domain clusters of three and four disulfide bridges. One of the bonds in the first cluster connects a central Cys residue of the NtD with a single conserved Cys residue in the linker. Site-directed mutagenesis of the Cys residue in the linker resulted in an endoplasmic reticulum precursor that was not matured and underwent a gradual degradation. The relevant disulfide bond has a crucial role in folding as it may stabilize the NtD and facilitate its interaction with the C-terminal portion of a Gas protein. The four disulfide bonds in the Cys-box are arranged in a manner consistent with a partial structural resemblance with the plant X8 domain, an independent carbohydrate-binding module that possesses only three disulfide bonds. Deletion of the Cys-box in Gas2 or Gas1 proteins led to the formation of an NtD devoid of any enzymatic activity. The results suggest that the Cys-box is required for proper folding of the NtD and/or substrate binding.     

Thioredoxins and glutaredoxins as facilitators of protein folding

Thiol-disulfide oxidoreductase systems of bacterial cytoplasm and eukaryotic cytosol favor reducing conditions and protein thiol groups, while bacterial periplasm and eukaryotic endoplasmatic reticulum provide oxidizing conditions and a machinery for disulfide bond formation in the secretory pathway. Oxidoreductases of the thioredoxin fold superfamily catalyze steps in oxidative protein folding via protein-protein interactions and covalent catalysis to act as chaperones and isomerases of disulfides to generate a native fold. The active site dithiol/disulfide of thioredoxin fold proteins is CXXC where variations of the residues inside the disulfide ring are known to increase the redox potential like in protein disulfide isomerases. In the catalytic mechanism thioredoxin fold proteins bind to target proteins through conserved backbone-backbone hydrogen bonds and induce conformational changes of the target disulfide followed by nucleophilic attack by the N-terminally located low pK(a) Cys residue. This generates a mixed disulfide covalent bond which subsequently is resolved by attack from the C-terminally located Cys residue. This review will focus on two members of the thioredoxin superfamily of proteins known to be crucial for maintaining a reduced intracellular redox state, thioredoxin and glutaredoxin, and their potential functions as facilitators and regulators of protein folding and chaperone activity.     

GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme

The native form of the bacterial glutathione transferase B1-1 (EC ) is characterized by one glutathione (GSH) molecule covalently linked to Cys-10. This peculiar disulfide, only found in the Beta and Omega class glutathione S-transferases (GSTs) but absent in all other GSTs, prompts questions about its role and how GSH can be activated and utilized in the reaction normally performed by GSTs. Stopped-flow and spectroscopic experiments suggest that, in the native enzyme (GSTB1-1ox), a second GSH molecule is present, albeit transiently, in the active site. This second GSH binds to the enzyme through a bimolecular interaction followed by a fast thiol-disulfide exchange with the covalently bound GSH. The apparent pK(a) of the non-covalently bound GSH is lowered from 9.0 to 6.4 +/- 0.2 in similar fashion to other GSTs. The reduced form of GSTB1-1 (GSTB1-1red) binds GSH 100-fold faster and also induces a more active deprotonation of the substrate with an apparent pK(a) of 5.2 +/- 0.1. Apparently, the absence of the mixed disulfide does not affect k(cat) and K(m) values in the GST conjugation activity, which is rate-limited by the chemical step both in GSTB1-1red and in GSTB1-1ox. However, GSTB1-1ox follows a steady-state random sequential mechanism whereas a rapid-equilibrium random sequential mechanism is adopted by GSTB1-1red. Remarkably, GSTB1-1ox and GSTB1-1red are equally able to catalyze a glutaredoxin-like catalysis using cysteine S-sulfate and hydroxyethyl disulfide as substrates. Cys-10 is an essential residue in this redox activity, and its replacement by alanine abolishes this enzymatic activity completely. It appears that GSTB1-1 behaves like an "intermediate enzyme" between the thiol-disulfide oxidoreductase and the GST superfamilies.     

Structural basis of hematopoietic prostaglandin D synthase activity elucidated by site-directed mutagenesis

Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH(2) to PGD(2) and conjugation of glutathione to 1-chloro-2, 4-dinitrobenzene. We introduced site-directed mutations of Tyr(8), Arg(14), Trp(104), Lys(112), Tyr(152), Cys(156), Lys(198), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K(m) for glutathione and PGH(2) in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K(m) for glutathione was increased. Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. These results indicate that Lys(112), Cys(156), and Lys(198) are involved in the binding of PGH(2); Trp(104) is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr(8) and Arg(14) are essential for activation of the thiol group of glutathione.     

Role of cysteine residues in the activity of rat glutathione transferase P (7-7): elucidation by oligonucleotide site-directed mutagenesis

To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) form (GST-P), a cDNA clone, pGP5, containing the entire coding sequence of GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 13, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JM109. All four Cys residues in rGST-P were independently substituted with alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards 1-chloro-2,4-dinitrobenzene as liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intra- or inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity.     

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.     

Kinetic characterization of native and cysteine 112-modified glutathione S-transferase A1-1: reassessment of nonsubstrate ligand binding

We have prepared human glutathione S-transferase isoform A1-1 (GST A1-1) which has been chemically modified at cysteine 112. These modifications include formation of mixed disulfides with glutathione ("glutathiolation") and cross-linkage of the GST dimer with bis-maleimides reacting with the equivalent Cys 112 residues of the two monomers. This residue (Cys 112) lies adjacent to the hydrophobic substrate binding site, and its side chain thiol projects into the large, solvent-filled cleft which is widely reported in the literature to be the binding site of nonsubstrate ligands. Both types of modification block this intersubunit cleft region and significantly change its chemical environment. Kinetic experiments with these altered enzymes revealed that neither type of modification affects the catalytic activity of GST A1-1 or the binding of nonsubstrate ligands. The lack of an effect on glutathione conjugation activity is somewhat surprising given the proximity of cysteine 112 to the hydrophobic substrate binding site. More surprising, however, is the observation that modification at cysteine 112 has no effect on the binding of nonsubstrate ligands. Furthermore, two of these ligands, lithocholic acid and estradiol disulfate, unexpectedly exhibited competitive inhibition of the unmodified enzyme, suggesting that they bind in the hydrophobic substrate site rather than some accessory ligand binding site. Together, these results strongly argue against the intersubunit cleft as the nonsubstrate ligand binding site and prompt a reassessment of how these ligands interact with GST A1-1.     

A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease.

Thioredoxin (Trx) from Escherichia coli was compared with bovine protein disulfide-isomerase (PDI) for its ability to catalyze native disulfide formation in either reduced or randomly oxidized (scrambled) ribonuclease A (RNase). On a molar basis, a 100-fold higher concentration of Trx than of PDI was required to give the same rate of native disulfide formation measured as recovery of RNase activity. A Pro-34 to His (P34H Trx) mutation in the active site of E. coli Trx (WCGPC), mimicking the two suggested active sites in PDI (WCGHC), increased the catalytic activity in disulfide formation about 10-fold. The mutant P34H Trx displayed a 35-mV higher redox potential (E'0) of the active site disulfide/dithiol relative to wild type Trx, making it more similar to the redox potential observed for PDI. This higher redox potential correlates well with the enhanced activity and suggests a role for the histidine side chain. Enzymatic isomerization of disulfides in scrambled, oxidized RNase requires the presence of a catalytic thiol such as GSH to initiate the thiol-disulfide interchange. Bovine thioredoxin reductase, together with NADPH, could replace GSH. For oxidative folding of reduced RNase in air with Trx, P34H Trx, or PDI, catalytic amounts of sodium selenite (1 microM) resulted in rapid disulfide formation and high yields of ribonuclease activity equivalent to previously known redox buffers of GSH and GSSG. These results demonstrate no obligatory role for glutathione in disulfide formation. A possible mechanism for the unknown thiol oxidative process accompanying folding and protein disulfide formation in vivo is discussed.