Interaction of a bisquaternary ammonium compound with the peripheral organophosphorus (POP) site on acetylcholinesterase

O-ethyl-S (2 diisopropylaminoethyl) methyl phosphorothiolate (MPT) is an active site-directed inhibitor of acetylcholinesterase (AChE). The inhibition of mouse muscle AChE by MPT as well as the inhibition of its individual molecular forms do not proceed as simple irreversible bimolecular reactions. The insolubilization of AChE into a semisolid matrix allows to characterize, after dialysis of all unbound ligand, a partially reversible phase of the inhibition by MPT. These results can be explained in terms of two different modes of inhibition by MPT: the classical irreversible phosphorylation of the active site and an inhibition phase involving the reversible binding of MPT at a site peripheral to the active site, the peripheral organophosphorus site (POP-site). We now find that BW 284 C 51, a reversible specific inhibitor of AChE which protects the active site against irreversible inhibition by low MPT concentrations, can prevent the occurrence of the partially reversible inhibition phase. Hence, BW may bind to a peripheral site that either overlaps or is linked to the POP-site.     

Influence of synthetic peptide inhibitors on the thermal stability of thermitase, a serine proteinase from Thermoactinomyces vulgaris

The influence of chloromethyl ketones and methyl ketones of N-acylated peptides on the thermal denaturation of thermitase was investigated in the presence and the absence of calcium ions. The chloromethyl ketone derivatives are known to react irreversibly with the enzyme, whereas the corresponding methyl ketones are reversible inhibitors. Both groups of inhibitors offer a broad variety of affinity constants. The irreversible inhibition of thermitase causes a marked stabilization against thermal denaturation. On the other hand, the enzyme stability is not influenced by the binding of reversible inhibitors. The stabilizing effect of calcium ions is not dependent on the inhibitor binding. The importance of bivalent interaction (bridge formation) in the active site region of the enzyme for its thermal stability is discussed.     

Characterization of two types of binding sites of substrate-like inhibitors in the heavy meromyosin molecule

The effects of several phosphorylating and alkylating analogs of the substrate on the ATPase activity of myosin and heavy meromyosin were compared. The data obtained confirmed the previously made assumption on the existence of two types of substrate-like inhibitor binding sites in the enzyme molecule. In one of the sites, presumably in the active one, there occurs a reversible competitive inhibition characterized by a high affinity for the inhibitors, which are mixed anhydrides of various mononucleotides and mesitylcarboxylic acid or its derivatives. An enhancement of hydrophobicity of these compounds causes an increase in their affinity for this site. At much higher concentrations of the inhibitors an irreversible inhibition takes place, the rate of inhibition being decreased with an increase in the phosphorylating capacity of the compound. This site possesses a far lower affinity for the inhibitors and reveals a certain specificity with respect to the analog mononucleotide moiety structure, i.e. a replacement of the 6-NH2-group by the 6-OH-group or an increase in the number of the phosphate residues result in a decrease of the efficiency of inhibition. No correlation between the analog capacity to cause irreversible inhibition and to act as an effective competitive inhibitor of reversible type has been shown to exist, thus allowing to use inhibitors of preferable action in one of the two types of the binding sites. No irreversible inhibition site was revealed when the ATPase activity of myosin subfragment I with and without the DTNB chains was investigated. Actin protects myosin against the inhibiting action of the analogs tested.     

Interaction of an organophosphate with a peripheral site on acetylcholinesterase

O-Ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (MPT) is an active site directed inhibitor of acetylcholinesterase (AChE). Inhibition of the Electrophorus electricus (G4) enzyme follows classical second-order kinetics. However, inhibition of total mouse skeletal muscle AChE and inhibition of the individual molecular forms from muscle, including the monomeric species, do not proceed as simple irreversible bimolecular reactions. Similarly, complex inhibition kinetics are observed for the purified enzyme from Torpedo californica. AChE can be cross-linked with glutaraldehyde into a semisolid matrix. Under these conditions the abnormal concentration dependence for MPT inhibition is accentuated, and a range of MPT concentrations can be found where inhibition of polymerized AChE is far less than that observed at lower concentrations. Inhibition in certain concentration ranges is partially reversible after removal of all unbound ligand. Thus, there are two different modes of organophosphorus inhibition by MPT: the classical irreversible phosphorylation of the active site and a reversible interaction at a site peripheral to the active center. Propidium, a well-studied peripheral site ligand, can prevent the later interaction. Hence, the second site of MPT interaction with AChE may overlap or be linked to the peripheral anionic site of AChE characterized by the binding of propidium and other peripheral site inhibitors.     

Exploring the S4 and S1 prime subsite specificities in caspase-3 with aza-peptide epoxide inhibitors

Caspase-3 is a prototypic executioner caspase that plays a central role in apoptosis. Aza-peptide epoxides are a novel class of irreversible inhibitors that are highly specific for clan CD cysteine proteases. The five crystal structures of caspase-3-aza-peptide epoxide inhibitor complexes reported here reveal the structural basis for the mechanism of inhibition and the specificities at the S1' and the S4 subsites. Unlike the clan CA cysteine proteases, the catalytic histidine in caspase-3 plays a critical role during protonation and subsequent ring opening of the epoxide moiety and facilitates the nucleophilic attack by the active site cysteine. The nucleophilic attack takes place on the C3 carbon atom of the epoxide and results in an irreversible alkylation of the active site cysteine residue. A favorable network of hydrogen bonds involving the oxyanion hole, catalytic histidine, and the atoms in the prime site of the inhibitor enhance the binding affinity and specificity of the aza-peptide epoxide inhibitors toward caspase-3. The studies also reveal that subtle movements of the N-terminal loop of the beta-subunit occur when the P4 Asp is replaced by a P4 Ile, whereas the N-terminal loop and the safety catch Asp179 are completely disordered when the P4 Asp is replaced by P4 Cbz group.     

Mechanistic studies on a novel, highly potent gold-phosphole inhibitor of human glutathione reductase

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When approximately 1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a K(d)((app)) for GSSG of 63 microm, and a k((obs)max) of 106 s(-1) at 4 degrees C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (K(d) = 0.46 microm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range.     

Development of a matrix-assisted laser desorption/ionization-mass spectrometry screening test to evidence reversible and irreversible inhibitors of CDC25 phosphatases

The cell division cycle 25 phosphatases (CDC25s) are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers, and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25A and -C inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS) to detect reversible inhibitors or submitted to peptide mass fingerprint (PMF) analysis to reveal irreversible inhibitors covalently bound to the protein active site. After its validation, the protocol is applied to the detection of a novel candidate inhibitor of CDC25s named SV37. The screening procedure, as well as the preliminary biological results, demonstrates that this compound behaves as a reversible inhibitor.     

Mechanism of selenium-glutathione peroxidase and its inhibition by mercaptocarboxylic acids and other mercaptans

In a systematic search for effectors of glutathione peroxidase, a number of mercaptocarboxylic acids and tertiary mercaptans were found to be strong and specific inhibitors of the enzyme glutathione peroxidase. Assessment of various models was made by linear and nonlinear least squares fitting techniques. The results support the formation of reversible enzyme-inhibitor complexes. The active site selenium is trapped by the rapid binding of the inhibitor in competition with GSH. Data are consistent with the formation of thioselenenate adducts of the active site. The kinetic model which best describes the observed inhibition by the very strong inhibitor mercaptosuccinate implies that a selenenic acid with a kinetically significant lifetime is not formed when hydroperoxide is reduced. A noncovalent binding site for GSH or the presence of a cysteine residue at the active site of the enzyme provides a mechanistic rationale for the observed kinetics. Three of the most potent inhibitors found in this study, mercaptosuccinate, penicillamine, and alpha-mercaptopropionylglycine, are currently used as slow-acting drugs in the treatment of rheumatoid arthritis. Overall, the evidence suggests that glutathione peroxidase may be involved in the etiology of this disease.     

Structure-reactivity probes for active site shapes of cholesterol esterase by carbamate inhibitors.

4,4'-Biphenyl-di-N-butylcarbamate (1), (S)-1,1'-bi-2-naphthyl-2, 2'-di-N-butylcarbamate (S-2), (S)-1, 1'-bi-2-naphthyl-2-N-butylcarbamate-2'-butyrate (S-3), 2, 2'-biphenyl-di-N-butylcarbamate (4), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-octylcarbamate (5), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-phenylcarbamate (6), 2, 2'-biphenyl-2-N-butylcarbamate-2'-butyrate (7), 2, 2'-biphenyl-2-N-butylcarbamate-2'-ol (8), 2, 2'-biphenyl-2-N-octylcarbamate-2'-ol (9), (R)-1, 1'-bi-2-N-naphthyl-2-butylcarbamate-2'-ol (R-10), and glyceryl-1,2, 3-tri-N-butylcarbamate (11) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase. All inhibitors are irreversible inhibitors of the enzyme. Carbamates 1-3 and 7-10 are the first alkyl chain and esteratic binding site-directed irreversible inhibitors due to the fact that the reactivity of the enzyme is protected by the irreversible inhibitor, trifluoroacetophenone in the presence of these carbamates. Carbamate 1 is the least potent inhibitor for the enzyme probably due to the fact that the inhibitor molecule adopts a linear conformation and one of the carbamyl groups of the inhibitor molecule covalently interacts with the first alkyl chain binding site of the enzyme while the other carbamyl group of the inhibitor molecule exposes outside the active site. With near orthogonal conformations at the pivot bond of biaryl groups, one carbamyl group of carbamates S-2, S-3, and R-10 covalently binds to the first alkyl chain binding site of the enzyme while the other carbamyl, butyryl, or hydroxy group can not bind covalently to the second alkyl chain binding site probably due to the orthogonal conformations. Carbamates 4-9 and 11 are very potent inhibitors for the enzyme probably due to the fact that all these molecules freely rotate at the pivot bond of the biphenyl or glyceryl group and therefore can fit well into the bent-shaped first and second alkyl chains binding sites of the enzyme. Although, carbamates 4-6 and 11 are irreversible inhibitors of cholesterol esterase, the enzyme is not protected but further inhibited by trifluoroacetophenone in the presence of these carbamates. Therefore, carbamates 4-6 and 11 covalently bind to the first alkyl chain binding site of the enzyme by one of the carbamyl groups and may also bind to the second alkyl chain binding site of the enzyme by the second carbamyl group. Besides the bent-shaped conformation, the inhibition by carbamate 6 is probably assisted by a favorable pi-pi interaction between Phe 324 at the second alkyl chain binding site of the enzyme and the phenyl group of the inhibitor molecule. For cholesterol esterase, carbamates 8-10 are more potent than carbamates S-2, 4, and 5 probably due to the fact that the inhibitor molecules interact with the second alkyl chain binding site of the enzyme through a hydrogen bond between the phenol hydroxy group of the inhibitor molecules and the His 435 residue in that site.     

Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing Acid amidase inhibition

The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA), with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 1-Isothiocyanatopentadecane (AM9023) was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-Benzyloxycarbonyl-L-serine β-lactone (N-Cbz-serine β-lactone), inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126) of the β-subunit, confirming a suggested mechanism of hNAAA inactivation by the β-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.     

Study of beta-glucosidase from Helix pomatia by active site-directed inhibitors

This work describes the purification of a beta-glucosidase (beta-D-glucoside-glucohydrolase EC 3.2.1.21) from the digestive juice of Helix pomatia and the study of the enzyme's active site by using different reversible and irreversible inhibitors. The catalytic constants of arylglycosides and their pH-dependent variations have also been determined. The inhibition studies demonstrate that conduritol epoxides are irreversible inhibitors of beta-glucosidase from the digestive juice of H. pomatia, and that nojirimicin shows tight binding with glucosidase: the formation and dissociation of the enzyme-inhibitor complex (dissociation constant 1.1 mumol/1) required several minutes.     

S-(4-Bromo-2,3-dioxobutyl)CoA: an affinity label for certain enzymes than bind acetyl-CoA.

S-(4-Bromo-2,3-dioxobutyl)-CoA, a potential affinity label for enzymes possessing a receptor site(s) for short-chain acyl-CoA, was synthesized by condensing CoA and 1,4-dibromo-2,3-butanedione in acidified methanol. The new reagent was tested as an active site-directed irreversible inhibitor with four enzymes that accept a short-chain acyl-CoA as substrate. With citrate synthase (pig heart) and acetyl-CoA hydrolase (beef kidney) irreversible inhibition was observed, and the rate of inactivation obeyed first-order kinetics. Benzoyl-CoA, a reversible competitive inhibitor versus acetyl-CoA with both citrate synthase and acetyl-CoA hydrolase, protected the active site of both enzymes against the irreversible inhibitor. The new reagent was an exceptionally potent irreversible inhibitor of acetoacetyl-CoA thiolase (beef liver). Relatively low concentrations of the reagent (≥1 μm) completely inhibited the thiolase in less than 2 min. Preincubation of thiolase with acetoacetyl-CoA protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. In contrast, irreversible inhibition of l-3-hydroxyacyl-CoA dehydrogenase (pig heart) was not observed. Instead, the new reagent appeared to be a weak alternate substrate for this dehydrogenase. In all cases, the new reagent exhibited tight reversible binding at the active site since the measured K_i's (and K_m) were in the range, 30 to 120 μm. It is anticipated that the new reagent will be suitable for investigating a number of acyl-CoA using enzymes by affinity labeling techniques.     

Evidence for hemiketals as intermediates in the inactivation of serine proteinases with halomethyl ketones

The mechanism of inactivation of serine proteinases by peptide halomethyl ketone inhibitors was studied through the inhibition of trypsin with a series of model peptide ketones (Lys-Ala-LysCH2X). In this series, X is a poor leaving group with increasing electron-withdrawing capacity (X = H, CH2CO2CH3, COCH3, OCOCH3, and F), and as expected, the peptide ketones are reversible, competitive inhibitors of trypsin. The strength of binding of these inhibitors to trypsin increases with the electron-withdrawing ability of X, indicating that the inhibition constant Ki obtained is a measure of reversible hemiketal formation between the inhibitor ketone carbonyl group and the hydroxyl group of the active site serine. A Hammett plot of -log Ki vs. sigma I, the inductive substituent constant of X, reveals a linear relationship between the free energy of binding and the electron-withdrawing power of X. The reversible binding constant obtained for the corresponding chloromethyl ketone Lys-Ala-LysCH2Cl falls on this line, indicating that the reversible binding involves hemiketal formation, which is followed by alkylation of the enzyme.     

Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416

U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.     

Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Incubation of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) results in the inactivation of the 3 beta-hydroxysteroid dehydrogenase enzyme activity following pseudo-first-order kinetics. A double-reciprocal plot of 1/kobs versus 1/[5'-FSBA] yields a straight line with a positive y intercept, indicative of reversible binding of the inhibitor prior to an irreversible inactivation reaction. The dissociation constant (Kd) for the initial reversible enzyme-inhibitor complex is estimated at 0.533 mM, with k2 = 0.22 min-1. The irreversible inactivation could be prevented by the presence of NAD+ during the incubation, indicating that 5'-FSBA inactivates the 3 beta-hydroxysteroid dehydrogenase activity by reacting at the NAD+ binding site. Although the enzyme was inactivated by incubation with 5'-FSBA, no incorporation of the inhibitor was found in labeling studies using 5'-[p-(fluorosulfonyl)benzoyl] [14C]adenosine. However, the inactivation of 3 beta-hydroxysteroid dehydrogenase activity caused by incubation with 5'-FSBA could be completely reversed by the addition of dithiothreitol. This indicates the presence of at least two cysteine residues at or in the vicinity of the NAD+ binding site, which may form a disulfide bond catalyzed by the presence of 5'-FSBA. The intramolecular cysteine disulfide bridge was found between the cysteine residues in the peptides 274EWGFCLDSR282 and 18IICLLVEEK26, by comparing the [14C]iodoacetic acid labeling before and after recovering the enzyme activity upon the addition of dithiothreitol.     

A double-headed cathepsin B inhibitor devoid of warhead

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range.     

Processing of the papain precursor. Purification of the zymogen and characterization of its mechanism of processing

The precursor of the cysteine protease papain has been expressed and secreted as propapain from insect cells infected with a recombinant baculovirus expressing a synthetic gene coding for prepropapain. This 39-kDa secreted propapain zymogen molecule is glycosylated and can be processed in vitro into an enzymatically active authentic papain molecule of 24.5 kDa (Vernet, T., Tessier, D.C., Richardson, C., Laliberté, F., Khouri, H. E., Bell, A. W., Storer, A. C., and Thomas, D. Y. (1990) J. Biol. Chem. 265, 16661-16666). Recombinant propapain was stabilized with Hg2+ and purified to homogeneity using affinity chromatography, gel filtration, and ion-exchange chromatographic procedures. The maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 65 degrees C and under reducing conditions. Precursor processing is inhibited by a variety of reversible and irreversible cysteine protease inhibitors but not by specific inhibitors of serine, metallo or acid proteases. Replacement by site-directed mutagenesis of the active site cysteine with a serine at position 25 also prevents processing. The inhibitor 125I-N-(2S,3S)-3-trans-hydroxycarbonyloxiran-2-carbonyl-L-tyrosine benzyl ester covalently labeled the wild type papain precursor, but not the C25S mutant, indicating that the active site is accessible to the inhibitor and is in a native conformation within the precursor. Based on biochemical and kinetic analyses of the activation and processing of propapain we have shown that the papain precursor is capable of autoproteolytic cleavage (intramolecular). Once free papain is released processing can then occur in trans (intermolecular).     

Inhibition of CYP2B4 by 2-ethynylnaphthalene: evidence for the co-binding of substrate and inhibitor within the active site

2-Ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components: (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.     

Irreversible inhibition of rat hepatic glutathione S-transferase isoenzymes by a series of structurally related quinones

The effect of several structurally related 1,4-benzoquinones (BQ) and 1,4-naphthoquinones (NQ) on the activity of rat hepatic glutathione S-transferases (GST) was studied. For the 1,4-benzoquinones, the extent of inhibition increased with an increasing number of halogen substituents. Neither the type of halogen nor the position of chlorine-atoms was of major importance. Similarly, 2,3-dichloro-NQ demonstrated a considerably higher inhibitory activity than 5-hydroxy-NQ. 2-Methyl derivatives of NQ did not inhibit GST activity at all. The irreversible nature of the inhibition was shown both by the time-course of the inhibition as well as by the fact that removal of the inhibitor by ultrafiltration did not restore the enzymatic activity. Incubation of quinones and enzyme in the presence of the competitive inhibitor S-hexyl-glutathione, slowed the inhibition considerably, indicating an involvement of the active site. Isoenzyme 3-3 was found to be most sensitive towards the whole series of inhibitors, whereas the activity of isoenzyme 2-2 was least affected in all cases. The inhibition by quinones is probably mainly due to covalent modification of a specific cysteine residue in or near the active site. The differential sensitivities of individual isoenzymes indicates that this residue is more accessible and/or easier modified in isoenzyme 3-3 than in any of the other isoenzymes tested. The findings suggest that quinones form a class of compounds from which a selective in vivo inhibitor of the GST might be developed.