Role of cardiac glutathione transferase and of the glutathione S-conjugate export system in biotransformation of 4-hydroxynonenal in the heart

There is a remarkable difference in the isozyme pattern between cardiac and hepatic glutathione S-transferases in rat (Ishikawa, T., and Sies, H. (1984) FEBS Lett. 169, 156-160), and one near-neutral isozyme (pI = 6.9) of the cardiac glutathione S-transferases was found to have a significantly high activity toward 4-hydroxynonenal. The isozyme was inhibited by the resulting glutathione S-conjugate of 4-hydroxynonenal competitively with GSH and noncompetitively with 4-hydroxynonenal. The kinetic parameters estimated for the isozyme were: kcat = 460 mol X min-1 X mol enzyme-1, Km = 50 microM for 4-hydroxynonenal, Ki = 85 microM. When the heart was perfused with 4-hydroxynonenal, a marked decrease was observed in the intracellular GSH level, accompanied by an increase of glutathione S-conjugate of 4-hydroxynonenal in the heart. The rate of the conjugation reaction was more than 30 times the rate of the spontaneous reaction, the half-life of 4-hydroxynonenal in the heart being less than 4 s. The glutathione S-conjugate of 4-hydroxynonenal was released from the heart into the perfusion medium. Saturation kinetics were observed for the release with respect to the intracellular level of the S-conjugate (Vmax = 12 nmol X min-1 X g heart-1), and there was a competition by the S-conjugate for GSSG release. The release of the glutathione S-conjugate is considered as a carrier-mediated process and to be important not only in interorgan glutathione metabolism but also in diminishing the inhibitory effect of the S-conjugate on glutathione S-transferases and glutathione reductase.     

Long chain lipid hydroperoxides increase the glutathione redox potential through glutathione peroxidase 4

BackgroundPeroxidation of PUFAs by a variety of endogenous and xenobiotic electrophiles is a recognized pathophysiological process that can lead to adverse health effects. Although secondary products generated from peroxidized PUFAs have been relatively well studied, the role of primary lipid hydroperoxides in mediating early intracellular oxidative events is not well understood.MethodsLive cell imaging was used to monitor changes in glutathione (GSH) oxidation in HAEC expressing the fluorogenic sensor roGFP during exposure to 9-hydroperoxy-10E,12Z-octadecadienoic acid (9-HpODE), a biologically important long chain lipid hydroperoxide, and its secondary product 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE). The role of hydrogen peroxide (H₂O₂) was examined by direct measurement and through catalase interventions. shRNA-mediated knockdown of glutathione peroxidase 4 (GPx4) was utilized to determine its involvement in the relay through which 9-HpODE initiates the oxidation of GSH.ResultsExposure to 9-HpODE caused a dose-dependent increase in GSH oxidation in HAEC that was independent of intracellular or extracellular H₂O₂ production and was exacerbated by NADPH depletion. GPx4 was involved in the initiation of GSH oxidation in HAEC by 9-HpODE, but not that induced by exposure to H₂O₂ or the low molecular weight alkyl tert-butyl hydroperoxide (TBH).ConclusionsLong chain lipid hydroperoxides can directly alter cytosolic E_GSH independent of secondary lipid oxidation products or H₂O₂ production. NADPH has a protective role against 9-HpODE induced E_GSH changes. GPx4 is involved specifically in the reduction of long-chain lipid hydroperoxides, leading to GSH oxidation.SignificanceThese results reveal a previously unrecognized consequence of lipid peroxidation, which may provide insight into disease states involving lipid peroxidation in their pathogenesis.     

4-Hydroxynonenal inhibits glutathione peroxidase: protection by glutathione.

4-Hydroxy-2,3-trans-nonenal, a lipid peroxidation product, inhibits glutathione peroxidase in a concentration-dependent manner. The concentration providing 50% inhibition is 0.12 mM. This inhibition can be almost completely (89%) prevented by 1 mM glutathione added to the incubation mixture 30 min before 4-hydroxy-2,3-trans-nonenal or 2,3-trans-nonenal, but not by other thiol-containing antioxidants such as 0.5 mM dithiothreitol or beta-mercaptoethanol. Again the addition of 1 mM glutathione, and not of 0.5 mM dithiothreitol or beta-mercaptoethanol, to the enzyme 30 min after incubation with 4-hydroxy-2,3-trans-nonenal restores activity to the same extent as does the preincubation with GSH. In view of the known reactivity of 4-hydroxy-2,3-trans-nonenal with lysine residues and the reversibility of the inhibition, the involvement of a lysine residue in GSH binding to glutathione peroxidase is proposed. The potential relevance of the inhibition of glutathione peroxidase by 4-hydroxy-nonenal to oxidative tissue damage is discussed with particular emphasis on neurological disorders.     

Formation of 4-ethoxy-4'-nitrosodiphenylamine in the reaction of the phenacetin metabolite 4-nitrosophenetol with glutathione

Phenacetin, a constituent of several analgesic and antipyretic formulations has been made responsible for a variety of toxic and carcinogenic actions. 4-Nitrosophenetol, the N-oxydation product of intermediate 4-phenetidine, forms methemoglobin and binds covalently to sulfhydryl groups of proteins and glutathione. In the reaction of 4-nitrosophenetol with glutathione and other thiols an intermediate so-called "semimercaptal" is formed from which N-(thiol-S-yl)-4-phenetidine S-oxide, N-(thiol-S-yl)-4-phenetidine and 4-phenetidine derive. Besides thiol adducts, a yellow compound is formed which was isolated as a pure crystalline product (elemental analysis) and identified by FAB-MS, EI-MS, 13C-, 1H-NMR, and UV-VIS spectroscopy as 4-ethoxy-4'-nitrosodiphenylamine. This nitrosoarene is formed by an unknown mechanism from 4-nitrosophenetol and 4-phenetidine under liberation of ethanol. In human erythrocytes this compound is easily reduced to 4-amino-4'-ethoxydiphenylamine (FAB-MS, EI-MS, 13C-NMR). During the reaction of 4-nitrosophenetol with red cells only traces of 4-ethoxy-4'-nitrosodiphenylamine were formed, whereas up to 10% appeared as the reduction product 4-amino-4'-ethoxydiphenylamine. This latter compound is unstable in red cells and is metabolized further to unidentified products.     

Specificity of the glutathione S-transferases in the conversion of leukotriene A4 to leukotriene C4

We have synthesized the 5,6-LTA4, 8,9-LTA4, and 14,15-LTA4 as methyl esters by an improved biomimetic method with yields as high as 70-80%. We have investigated the catalytic efficiency of the purified cytosolic glutathione S-transferase (GST) isozymes from rat liver in the conversion of these leukotriene epoxides to their corresponding LTC4 methyl esters. Among various rat liver GST isozymes, the anionic isozyme, a homodimer of Yb subunit, exhibited the highest specific activity. In general, the isozymes containing the Yb subunit showed better activity than the isozymes containing the Ya and/or Yc subunits. Interestingly, all three different LTA4 methyl esters gave comparable specific activities with a given GST isozyme indicating that regiospecificity of GSTs was not the factor in determining their ability to catalyze this reaction. Surprisingly, purified GSTs from sheep lung and seminal vesicles showed little activity toward these leukotriene epoxides, indicating a lack of the counterpart of rat liver anionic GST isozyme in these tissues.     

Dynamics of glutathione conjugation and conjugate efflux in detoxification of the carcinogen, 4-nitroquinoline 1-oxide: contributions of glutathione, glutathione S-transferase, and MRP1

4-Nitroquinoline 1-oxide (NQO) is a reactive electrophile with potent cytotoxic as well as genotoxic activities. NQO forms a conjugate, QO-SG, with glutathione, which greatly reduces its chemical reactivity. Previous studies demonstrated that glutathione S-transferase (GST) P1a-1a and multidrug resistance protein (MRP) 1/2 act in synergy to confer resistance to both cyto- and genotoxicities of NQO, whereas protection afforded by GSTP1a-1a or MRP alone was much less. To better understand the role of glutathione, GSTP1a-1a, and MRP1 in NQO detoxification, we have characterized the kinetics and cofactor requirements of MRP1-mediated transport of QO-SG and NQO. Additionally, using recombinant GSTP1a-1a and physiological conditions, we have examined the enzymatic and nonenzymatic formation of QO-SG. Results show that MRP1 supports efficient transport of QO-SG with a K(m) of 9.5 microM and a V(max) comparable to other good MRP1 substrates. Glutathione or its S-methyl analogue enhanced the rate of (3)H-QO-SG transport, whereas QO-SG inhibited the rate of (3)H-glutathione transport. These data favor a mechanism for glutathione-enhanced, MRP1-mediated QO-SG transport that does not involve cotransport of glutathione. NQO was not transported by MRP1 either alone or in the presence of S-methyl glutathione. Transport of (3)H-NQO was observed in the presence of glutathione, but uptake into MRP1-containing vesicles was entirely attributable to its conjugate, QO-SG, formed nonenzymatically. While the nonenzymatic rate was readily measurable, enzyme catalysis was overwhelmingly dominant in the presence of GSTP1a-1a (rate enhancement factor, (k(cat)/K(m))/k(2), approximately 3 x 10(6)). We conclude that MRP1 supports detoxification of NQO via efficient, glutathione-stimulated efflux of QO-SG. While nonenzymatic QO-SG formation and MRP1-mediated conjugate efflux result in low-level protection from cyto- and genotoxicities, this protection is greatly enhanced by coexpression of GSTP1-1 with MRP1. This result emphasizes the quantitative importance of enzyme-catalyzed conjugate formation, a crucial determinant of high-level, MRP-dependent protection of cells from NQO toxicity.     

S-(4-Bromo-2,3-dioxobutyl)glutathione: a new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase

S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 microM) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The kobs exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 microM, with a kmax of 0.078 min-1 and K1 = 66 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, completely protects against inactivation by S-BDB-G. About 1.3 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated concomitant with 100% inactivation, whereas only 0.48 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when activity is fully retained. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH4, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified: Lys82-His-Asn-Leu-X-Gly-Glu-Thr-Glu-Glu-Glu-Arg93, in which X is modified Cys86, and Leu109-Gln-Leu-Ala-Met-CmCys-Y-Ser-Pro-Asp-Phe-Glu-Arg121 , in which Y is modified Tyr115. Only the Lys82-Arg93 peptide was modified in the presence of S-hexylglutathione when the enzyme retained full activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.     

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.     

Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes

mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.     

Crystal structure of a murine glutathione S-transferase in complex with a glutathione conjugate of 4-hydroxynon-2-enal in one subunit and glutathione in the other: evidence of signaling across the dimer interface.

mGSTA4-4, a murine glutathione S-transferase (GST) exhibiting high activity in conjugating the lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) with glutathione (GSH), was crystallized in complex with the GSH conjugate of 4-HNE (GS-Hna). The structure has been solved at 2.6 A resolution, which reveals that the active site of one subunit of the dimeric enzyme binds GS-Hna, whereas the other binds GSH. A marked asymmetry between the two subunits is evident. Most noticeable are the differences in the conformation of arginine residues 69 and 15. In all GST structures published previously, the guanidino groups of R69 residues from both subunits stack at the dimer interface and are related by a (pseudo-) 2-fold axis. In the present structure of mGSTA4-4, however, the two R69 side chains point in opposite directions, although their guanidino groups remain in contact. In the subunit with bound GSH, R69 also interacts with R15, and the guanidino group of R15 points away from the active site, whereas in the subunit that binds GS-Hna, R15 pivots into the active site, which breaks its interaction with R69. According to our previous results [Nanduri et al. (1997) Arch. Biochem. Biophys. 335, 305-310], the availability of R15 in the active site assists the conjugation of 4-HNE with GSH. We propose a model for the catalytic mechanism of mGSTA4-4 in conjugating 4-HNE with GSH-i.e., the guanidino group of R15 is available in the active site of only one subunit at any given time and the stacked pair of R69 residues act as a switch that couples the concerted movement of the two R15 side chains. The alternate occupancy of 4-HNE in the two subunits has been confirmed by our kinetic analysis that shows the negative cooperativity of mGSTA4-4 for 4-HNE. Disruption of the signaling between the subunits by mutating the R69 residues released the negative cooperativity with 4-HNE.     

Regulation of 4-hydroxynonenal-mediated signaling by glutathione S-transferases

4-Hydroxynonenal (HNE), one of the major end products of lipid peroxidation, has been shown to be involved in signal transduction and available evidence suggests that it can affect cell cycle events in a concentration-dependent manner. Glutathione S-transferases (GSTs) can modulate the intracellular concentrations of HNE by affecting its generation during lipid peroxidation by reducing hydroperoxides and also by converting it into a glutathione conjugate. We have recently demonstrated that overexpression of the Alpha class GSTs in cells leads to lower steady-state levels of HNE, and these cells acquire resistance to apoptosis induced by lipid peroxidation-causing agents such as H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics, suggesting that signaling for apoptosis by these agents is transduced through HNE. Cells with the capacity to exclude HNE from the intracellular environment at a faster rate are relatively more resistant to apoptosis caused by H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics as well as by HNE, suggesting that HNE may be a common denominator in mechanisms of apoptosis caused by oxidative stress. We have also shown that transfection of adherent cells with HNE-metabolizing GSTs leads to transformation of these cells due to depletion of HNE. These recent studies from our laboratories, which strongly suggest that HNE is a key signaling molecule and that GSTs, being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.     

Measurement of oxidized glutathione and total glutathione in the perfused rat heart

1. A method was developed for the assay of GSSG in heart tissue. 2. GSSG and total glutathione were measured in rat hearts perfused under a variety of conditions. About 2% of the total glutathione is present as GSSG. The concentrations of GSSG and GSH remained constant under all the conditions tested. 3. These results are discussed with reference to the equilibrium and rate of the glutathione reductase reaction in the cell. It is concluded that the enzyme reaction does not lie near equilibrium.     

Dopamine quinone modifies and decreases the abundance of the mitochondrial selenoprotein glutathione peroxidase 4

Oxidative stress and mitochondrial dysfunction are known to contribute to the pathogenesis of Parkinson's disease. Dopaminergic neurons may be more sensitive to these stressors because they contain dopamine (DA), a molecule that oxidizes to the electrophilic dopamine quinone (DAQ) which can covalently bind nucleophilic amino acid residues such as cysteine. The identification of proteins that are sensitive to covalent modification and functional alteration by DAQ is of great interest. We have hypothesized that selenoproteins, which contain a highly nucleophilic selenocysteine residue and often play vital roles in the maintenance of neuronal viability, are likely targets for the DAQ. Here we report the findings of our studies on the effect of DA oxidation and DAQ on the mitochondrial antioxidant selenoprotein glutathione peroxidase 4 (GPx4). Purified GPx4 could be covalently modified by DAQ, and the addition of DAQ to rat testes lysate resulted in dose-dependent decreases in GPx4 activity and monomeric protein levels. Exposing intact rat brain mitochondria to DAQ resulted in similar decreases in GPx4 activity and monomeric protein levels as well as detection of multiple forms of DA-conjugated GPx4 protein. Evidence of both GPx4 degradation and polymerization was observed following DAQ exposure. Finally, we observed a dose-dependent loss of mitochondrial GPx4 in differentiated PC12 cells treated with dopamine. Our findings suggest that a decrease in mitochondrial GPx4 monomer and a functional loss of activity may be a contributing factor to the vulnerability of dopaminergic neurons in Parkinson's disease.     

Dissection of the catalytic mechanism of isozyme 4-4 of glutathione S-transferase with alternative substrates

The kinetic and chemical mechanism of isozyme 4-4 of rat liver glutathione (GSH) S-transferase was investigated by using several alternative peptide substrates including N-acetyl-GSH, gamma-L-glutamyl-L-cysteine (gamma-GluCys), N4-(malonyl-D-cysteinyl)-L-2,4-diaminobutyrate (retro-GSH), and N4-(N-acetyl-D-cysteinyl)-L-2,4-diaminobutyrate (decarboxylated retro-GSH). The enzyme, which is normally stereospecific in the addition of GSH to the oxirane carbon of R absolute configuration in arene oxide substrates, loses its stereospecificity toward phenanthrene 9,10-oxide with the retro peptide analogues, giving a 2:1 mixture of the S,S and R,R stereoisomeric 9,10-dihydro-9-(S-peptidyl)-10-hydroxyphenanthrenes. The analogues with normal peptide bonds (N-acetyl-GSH and gamma-GluCys) show normal stereospecific addition. The kinetic mechanism of the enzyme was investigated by using the alternative substrate diagnostic with several 4-substituted 1-chloro-2-nitrobenzenes and GSH, N-acetyl-GSH, and gamma-GluCys. Varying the concentration of electrophile vs the identity of the GSH analogue and the concentration of GSH vs the identity of the electrophile gave two sets of intersecting reciprocal plots, a result consistent with a random sequential kinetic mechanism. The pH profiles of kc and kc/Ksm [saturating GSH, variable 1-chloro-2,4-dinitrobenzene (1)] exhibit a dependence on a deprotonation in the enzyme-GSH-1 and enzyme-GSH complexes with molecular pKa's of 6.1 and 6.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular implications of the human glutathione transferase A-4 gene (hGSTA4) polymorphisms in neurodegenerative diseases

Several lines of evidence, including an increased level of lipid peroxidation and the depletion of antioxidant molecules like as glutathione (GSH), indicate that oxidative stress plays an important role in the pathogenesis of several neurodegenerative disorders, such as Parkinson's disease (PD) and Alzheimer's disease (AD). We previously observed a significant increased level of DNA oxidative damage in peripheral blood cells of PD patients, with respect to controls, moreover, the activity of glutathione transferases (GSTs) measured in circulating plasma was higher in controls than in PD patients, suggesting a lower enzymatic protection in PD individuals. Among human GSTs, glutathione transferase A4-4 displays a high catalitic activity towards 4-hydroxy-2-nonenal (HNE), a marker of lipid peroxidation whose levels have been found significantly increased in the substantia nigra of Parkinson's disease patients, in respect to controls. We performed this study to determine the presence of allelic variants of functional interest in the coding region of the hGSTA4 gene on 60 PD patients and 60 healthy controls. By the combined effort of polymerase chain reaction/single-strand conformation polymorphisms (PCR/SSCP) techniques, we observed a single nucleotide polymorphism (SNP) G351A leading to the silent mutation Gln117Gln. No significant difference was observed in the distribution of this polymorphism between PD individuals and controls, moreover, we did not observe any other polymorphism in the hGSTA4 gene in our population. Further studies are required to test the role played by both factors regulating the level of the expression of the hGSTA4 gene and any possible post-translational modification of the protein, in the protection against oxidative damage in neuronal cells.     

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.