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.     

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.     

Differential expression of glutathione S-transferase isoenzymes in murine small intestine and colon

Glutathione (GSH) S-transferase (GST) isoenzymes of the small intestine and colon of female A/J mice have been purified and characterized to determine their interrelationships with other murine GSTs. Cytosolic GST activity in the small intestine was at least due to six isoenzymes with isoelectric points (pI) of 9.5, 9.3, 9.1, 8.5, 6.2 and 5.5. Small intestine isoenzymes with pI values of 9.5, 9.3, 8.5, and 6.2 were identical to the mGSTA1-1 (Alpha class), mGSTP1-1 (Pi class), mGSTM1-1 (Mu class) and mGSTA4-4 (Alpha class), respectively, of other A/J mouse tissues on the basis of their reverse-phase HPLC elution profile, immunological cross-reactivity and/or N-terminal region amino acid sequence. Even though GST9.1 of the small intestine cross-reacted with the antibodies raised against Pi class GST, reverse-phase HPLC and N-terminal amino acid sequence analyses suggested that this isoenzyme may be structurally different from mGSTP1-1 as well as mGSTP2-2. Likewise, despite immunological similarity with the Mu class GSTs, small intestine GST5.5 appeared to be different from other Mu class murine GSTs characterized previously. Cytosolic GST activity in the colon was mainly due to four isoenzymes with pI values of 9.8, 9.4, 6.6 and 5.8. While the identity of colon GST6.6 could not be established due to its low abundance, GST9.8, GST9.4 and GST5.8 were identical to mGSTP1-1, mGSTM1-1 and mGSTA4-4, respectively, of other A/J mouse tissues including the small intestine. Isoenzymes corresponding to small intestine GST9.1 and GST5.5 could not be detected in the colon. The results of the present study indicate that the small intestine of female A/J mice is better equipped for protection against toxic effects of electrophiles than colon.     

Glutathione transferase isoenzymes from human prostate.

By using affinity-chromatography and isoelectric-focusing techniques, several forms of glutathione transferase (GSTs) were resolved from human prostate cytosol. All the three major classes of GST, i.e. Alpha, Mu and Pi, are present in human prostate. However, large inter-individual variation in the qualitative and quantitative expression of different isoenzymes resulted in the samples investigated. The most abundant group of prostate isoenzymes showed acid (pI 4.3-4.7) behaviour and were classified as Pi class GSTs on the basis of their immunological and structural properties. Immunohistochemical staining of Pi class GSTs was prevalently distributed in the epithelial cells surrounding the alveolar lumen. Class Mu GSTs are also expressed, although in small amounts and in a limited number of samples, by human prostate. The major cationic isoenzyme purified from prostate, GST-9.6; (pI 9.6; apparent subunit molecular mass of 28 kDa), appears to be different from the cationic GST alpha-epsilon forms isolated from human liver and kidney as evidenced by its structural, kinetical and immunological properties. This enzyme, which accounts for about 20-30% (on protein basis) of total amount of GSTs, is expressed by only 40% of samples. GST-9.6 has the ability to cross-react in immunoblotting analysis with antisera raised against rat liver GST 2-2, rather than with antisera raised against members of human Alpha, Mu and Pi class GSTs. Although prostate GST-9.6 shows close relationship with the human skin GST pI 9.9, it does not correspond to any other known human GST.     

Engineering a new C-terminal tail in the H-site of human glutathione transferase P1-1: structural and functional consequences

We have sought the structural basis for the differing substrate specificities of human glutathione transferase P1-1 (class Pi) and human glutathione transferase A1-1 (class Alpha) by adding an extra helix (helix 9), found in the electrophilic substrate-binding site (H-site) of the human class Alpha enzyme, at the C terminus of the human class Pi enzyme. This class Pi-chimera (CODA) was expressed in Escherichia coli, purified and characterized by kinetic and crystallographic approaches. The presence of the newly engineered tail in the H-site of the human Pi enzyme alters its catalytic properties towards those exhibited by the human Alpha enzyme, as assessed using cumene hydroperoxide (diagnostic for class Alpha enzymes) and ethacrynic acid (diagnostic for class Pi) as co-substrates. There is a change of substrate selectivity in the latter case, as the k(cat)/K(m)(EA) value decreases about 70-fold, compared to that of class Pi. With 1-chloro-2,4-dinitrobenzene as co-substrate there is a loss of catalytic activity to about 2% with respect to that of the Pi enzyme. Crystallographic and kinetic studies of the class Pi-chimera provide important clues to explain these altered catalytic properties. The new helix forms many complimentary interactions with the rest of the protein and re-models the original electrophilic substrate-binding site towards one that is more enclosed, albeit flexible. Of particular note are the interactions between Glu205 of the new tail and the catalytic residues, Tyr7 and Tyr108, and the thiol moiety of glutathione (GSH). These interactions may provide an explanation of the more than one unit increase in the pK(a) value of the GSH thiolate and affect both the turnover number and GSH binding, using 1-chloro-2,4-dinitrobenzene as co-substrate. The data presented are consistent with the engineered tail adopting a highly mobile or disordered state in the apo form of the enzyme.     

Cytosolic rat liver glutathione transferase 4-4. Primary structure of the protein reveals extensive differences between homologous glutathione transferases of classes alpha and mu

The primary structure of the class Mu glutathione transferase 4-4 from rat liver was determined. The structural data characterize a class Mu protein within an enzyme family for which three classes have been distinguished (Alpha, Mu, Pi). The structure was determined by analysis of peptides obtained after treatment with trypsin. Glu-specific protease and CNBr. The protein is composed of two identical subunits, each with 217 amino acid residues. No evidence for microheterogeneity or for the presence of modified residues was encountered. The primary structure was found to be strictly homologous with corresponding parts in known regions of other class Mu enzymes of rat, mouse, human and bovine origin. Relationships to the cytosolic enzyme of other classes (Pi and Alpha) are considerably more distant. A comparison with the entire chain of the class Alpha subunit 1 from rat liver was carried out by three methods, alignment of amino acid sequences, correlation of hydrophilicity plots and predictions of secondary structures. All methods reveal weak similarities but also large differences. The overall positional identity is only 26%. Combined, the results establish the first complete class Mu structure, show distant inter-class relationships, and relate subunit 4 (class Mu) and subunit 1 (class Alpha) in a family of enzymes rather than in a group of isoenzymes.     

Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines

Enzyme-linked immunoassays (ELISAs) based on the double-antibody sandwich technique have been developed for the quantitative analysis of the major human cytosolic class Pi, Mu and Alpha glutathione transferases (GSTs). The procedures were optimized with respect to antibody concentration for coating of plates as well as other parameters in order to achieve high sensitivity and accuracy. No cross-reactivity was detected between members of the three different classes of GSTs or among the Mu class GSTs M2-2, M3-3 and M4-4 with the ELISA for GST M1-1. The ELISAs have been applied to establish the cytosolic GST profiles of 10 cell lines and to monitor the plasma GST levels in cancer patients. The results revealed that the class Pi GST was the dominant isoenzyme in six (LS 174T, HCT-8, Hu 549 Pat, K-562, U-937 and Hu 549) out of nine tumor cell lines and immortalized hepatocytes (Chang Liver). The isoenzymes A1-1 and M1-1 were determined to be the major GST components in Hep G2 and HeLa cells, respectively. In a clinical study, the majority of the patients with urinary bladder cancer were found to have increased plasma levels of both GST A1-1 and GST P1-1 (10/15), while patients with renal cancer frequently showed increases only in GST P1-1 (5/8). The results demonstrate that the ELISAs are suitable for analyzing GST phenotypes in both normal and tumor cells and in monitoring plasma levels of GSTs in cancer patients.     

Ab initio calculations on hidden modulators of theta class glutathione transferase activity

The glutathione transferases decrease the pKa of glutathione, allowing its deprotonation and the formation of the more reactive thiolate anion. The thiolate is maintained in the active site through a weak conventional hydrogen bond first sphere interaction donated by a Tyr hydroxyl in the Alpha, Mu, Pi, and Sigma glutathione transferase classes that can be modified by other second sphere or indirect thiolate contacts. However, the Theta and Delta class isoforms use a Ser hydroxyl for stabilizing the GSH thiolate, and as such, have a different chemical system compared with that of the Tyr possessed by other classes. We have used high level ab initio methods to investigate this interaction by using a simple methanol methanethiol system as a model. The hydrogen bond strength of this initial first sphere interaction was calculated to be less than that of the Tyr interaction. A putative second sphere interaction exists in the Theta and Delta class structures between Cys or Ser-14 and Ser-11 in the mammalian Theta subclass 1 and 2, respectively. The effect of this interaction on the first sphere interaction has also been investigated and found to significantly increase the energy of the bond.     

Glutathione transferase superfamily behaves like storage proteins for dinitrosyl-diglutathionyl-iron complex in heterogeneous systems

Electron paramagnetic resonance and kinetics experiments have been made to determine the formation, stability, and fate of the natural nitric oxide carrier, dinitrosyl-diglutathionyl-iron complex (DNDGIC), in heterogeneous systems approaching in vivo conditions. Both in human placenta and rat liver homogenates DNDGIC is formed spontaneously from GSH, S-nitroso-glutathione, and trace amounts of ferrous ions. DNDGIC is unstable in homogenates depleted of glutathione S-transferase (GST); an initial phase of rapid decomposition is followed by a slower decay, which is inversely proportional to the concentration. In the crude human placenta homogenate, GSTP1-1, which represents 90% of the cytosolic GST isoenzymes, is the preferential target for DNDGIC. It binds the complex almost stoichiometrically and stabilizes it for several hours (t1/2 = 8 h). In the presence of an excess of DNDGIC, negative cooperativity in GSTP1-1 opposes the complete loss of the usual detoxicating activity of this enzyme. In the rat liver homogenate, multiple endogenous GSTs (mainly Alpha and Mu class isoenzymes) bind the complex quantitatively and stabilize it (t1/2 = 4.5 h); negative cooperativity is also seen for these GSTs. Thus, the entire pool of cytosolic GSTs, with the exception of the Theta GST, represents a target for stoichiometric amounts of DNDGIC and may act as storage proteins for nitric oxide. These results confirm the existence of a cross-link between NO metabolism and the GST superfamily.     

Substrate specificity of rat liver glutathione S-transferase isoenzymes for a series of glutathione analogues, modified at the gamma-glutamyl moiety.

The substrate specificity of purified rat liver glutathione S-transferases (GSTs) for a series of gamma-glutamyl-modified GSH analogues was investigated. GST isoenzyme 3-3 catalysed the conjugation of 1-chloro-2,4-dinitrobenzene with six out of the nine analogues. alpha-L-Glu-L-Cys-Gly and alpha-D-Glu-L-Cys-Gly showed catalytic efficiencies of 40% and 130% that of GSH respectively. The GSH analogue with an alpha-D-glutamyl moiety appeared to be a highly isoenzyme-3-3-specific co-substrate: kcat./Km with GST isoenzyme 4-4 was only about 5% that with GST isoenzyme 3-3, and no enzymic activity was detectable with GST isoenzymes 1-1 and 2-2. GST isoenzyme 4-4 showed some resemblance to GST 3-3: five out of nine co-substrate analogues were accepted by this second isoenzyme of the Mu multigene family. Isoenzymes 1-1 and 2-2, of the Alpha multigene family, accepted only two alternative co-substrates, which indicates that their GSH-binding site is much more specific.     

Quantification of human hepatic glutathione S-transferases.

Human hepatic glutathione S-transferase (GST) subunits were characterized and quantified with the aid of a recently developed h.p.l.c. method. In 20 hepatic tissue specimens the absolute amounts of the basic Class Alpha subunits B1 and B2, the near-neutral Class Mu subunits mu and psi and the acidic subunit pi were determined. The average total amount of GST was 37 micrograms/mg of cytosolic protein, with the Class Alpha GST being the predominant class (84% of total GSTs), and pi as the sole representative of the Class Pi GSTs present in the lowest concentration (4% of total GSTs). Large interindividual differences were observed for all subunits, with variations up to 27-fold, depending on the subunit. For the Class Alpha GST-subunits B1 and B2, a biphasic ratio was observed. The genetic polymorphism of the subunits mu and psi was confirmed by h.p.l.c. analysis, and correlated with the enzymic glutathione conjugation of trans-stilbene oxide and with Western blotting of cytosols, using a monoclonal anti-(Class Mu GST) antibody. Of the 20 livers examined, ten contained only mu, whereas the occurrence of psi alone, and the combination of mu and psi, were found in only one liver each.     

Differential expression of Alpha,Mu, and Pi classes of glutathione S-transferases in chemosensory mucosae of rats during development

The expression of three classes of glutathione S-transferases (GSTs), Alpha, Mu, and Pi was investigated in the nasal mucosae of rats during development using immunohistochemical methods. GST Alpha and Mu were first detected in the supranuclear region of sustentacular cells on embryonic days 16. The Bowman's glands expressed differential patterns of immunoreactivity during development, beginning at postnatal day (P) 2 and P6 for Alpha and Mu classes, respectively and being greatest at P11 for both. The acinar cells of vomeronasal glands in the vomeronasal organ expressed Alpha and Mu classes of GSTs from P11 onwards. In the septal organ of Masera, the supranuclear region of sustentacular cells expressed GSTs from P11 with little or no variation during development. In the respiratory mucosa, Alpha and Mu classes of GSTs were detected at the brush borders of ciliated cells and in the acinar cells of posterior septal glands, but not in anterior septal or respiratory glands located on the turbinates. Compared to olfactory mucosa, the changes in immunoreactivity for GSTs were less pronounced in the respiratory mucosa during development. Specific GST Pi immunoreactivity was not detected in the nasal mucosae at any stage of development studied. The occurrence of GSTs in the nasal mucosa, including olfactory, vomeronasal, septal, and respiratory epithelia, suggests that the GSTs are actively involved in the biotransformation of xenobiotics including odorants and pheromones, and may also participate in perireceptor processes such as odorant clearance. In addition, we have developed a working model describing the cellular localization of certain phase I (e.g., cytochrome P-450s) and phase II (e.g., GSTs, -glutamyl transpeptidase) biotransformation enzymes in the olfactory mucosa and their proposed roles in xenobiotic metabolism.     

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.     

Nitrosylation of human glutathione transferase P1-1 with dinitrosyl diglutathionyl iron complex in vitro and in vivo

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1.     

Mouse hepatic glutathione transferase isoenzymes and their differential induction by anticarcinogens. Specificities of butylated hydroxyanisole and bisethylxanthogen as inducers of glutathione transferases in male and female CD-1 mice.

GSH transferase isoenzymes of class Mu (two forms), class Pi (one form) and class Alpha (two forms) were purified from liver cytosols of female CD-1 mice pretreated with an anticarcinogenic inducer, 2(3)-t-butyl-4-hydroxyanisole. GSH transferases GT-8.7, GT-8.8a and GT-8.8b, GT-9.0, GT-9.3, GT-10.3 and GT-10.6 contained a minimum of six types of subunits distinguishable by structural, catalytic and immunological characteristics. H.p.l.c. analysis of the subunit compositions of affinity-purified GSH transferases from liver cytosols of induced and non-induced male and female CD-1 mice showed that two anticarcinogenic compounds, 2(3)-t-butyl-4-hydroxyanisole and bisethylxanthogen, differed markedly in their specificities as inducers of GSH transferase.     

A highly acidic tyrosine 9 and a normally titrating tyrosine 212 contribute to the catalytic mechanism of human glutathione transferase A4-4

Human glutathione transferase A4-4 is an enzyme catalyzing the detoxication of intracellularly produced electrophiles such as 4-hydroxynonenal and other alkenal products of lipid peroxidation. Two tyrosines in the active site of the enzyme have been studied with help of UV difference spectroscopy and site-directed mutagenesis. The titration curve of GST A4-4 shows a pK(a) of 6.7 attributable to tyrosine 9, which in the Y212F mutant was shifted to pK(a) 7.1. In both cases the pK(a) was independent of the absence or presence of GSH. Thus, the active-site tyrosine 9 of this isoenzyme is more than one unit more acidic than the corresponding tyrosine of other Alpha class glutathione transferases. The tyrosines remaining in the Y9F mutant titrate like free tyrosine with pK(a) values > or = 10. A mechanism involving a tyrosine-9-bound water molecule acting as a proton shuttle is proposed for the Michael additions catalyzed by GST A4-4.     

Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate

Summary. Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-γ-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC₅₀=0.11±0.01 μM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-γ-(γ-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the γ-glutamyl-cysteine peptide bond, has been developed as γ-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the γ-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.     

Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class Alpha glutathione transferase

Human class Alpha glutathione transferase (GST) A1-1 has been subjected to site-directed mutagenesis of a Tyr residue conserved in all classes of cytosolic GSTs. The change of Tyr8----Phe lowers the specific activities with three substrates to 2-8% of the values for the wild-type enzyme. The changes in the kinetic parameters kcat/KM, Vmax and S0.5 show that the decreased activities are partly due to a reduced affinity for glutathione. The effect is reflected in lowered kcat values, suggesting that the hydroxyl group of Tyr8 is involved in the activation of glutathione. The proposal of such a role for the Tyr residue has support from the 3D structure of a pig lung class Pi GST [Reinemer et al. (1991) EMBO J. 10, 1997-2005]. Thus, Tyr8 appears to be the first active site residue established as participating in the chemical mechanism of a GST.     

Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions

GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.     

Proteomic identification of glutathione S-transferases from the model nematode Caenorhabditis elegans

Glutathione affinity chromatography and two-dimensional electrophoresis (2-DE) were used to purify glutathione binding proteins from Caenorhabditis elegans. All proteins identified after peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight were found to belong to the glutathione S-transferase (GST) superfamily. From the 26 individual spots identified, 12 different GSTs were isolated. Of these, five were found on the gel only once, whilst the remaining seven were represented by 21 separate spots. Most of the GSTs identified were of the nematode specific class, however, three Alpha class GSTs, a Pi and a Sigma class GST were also isolated.