Peroxidase-mediated conjugation of glutathione to unsaturated phenylpropanoids. Evidence against glutathione S-transferase involvement

A number of plant species are thought to possess a glutathione S-transferase enzyme (GST: EC 2.5.1.18) that will conjugate glutathione (GSH) to trans -cinnamic acid (CA) and para -coumaric acid (4-CA). However, we present evidence that this activity is mediated by peroxidase enzymes and not GSTs. The N-terminal amino acid sequence of the GSH-conjugating enzyme purified from etiolated corn shoots exhibited a strong degree of homology to cytosolic ascorbate peroxidase enzymes (APX: EC 1.11.1.11) from a number of plant species. The GSH-conjugating and APX activities of corn could not be separated during chromatography on hydrophobic-interaction. anion-exchange, and gel filtration columns. Spectral analysis of the enzyme revealed that the protein had a Soret band at 405 nm. When the enzyme was reduced with dithionite, the peak was shifted to 423 nm with an additional peak at 554 nm. The spectrum of the dithionite-reduced enzyme in the presence of 0.1 m M KCN exhibited peaks at 430, 534 and 563 nm. These spectra are consistent with the presence of a heme moiety. The GSH-conjugating and APX activities of the enzyme were both inhibited by KCN. NaN₃, p -chloromercuribenzoate ( p CMB), and iodoacetate. The APX specific activity of the enzyme was 1.5-fold greater than the GSH-conjugating specific activity with 4-CA. In addition to the corn enzyme, a pea recombinant APX (rAPX) and horseradish peroxidase (HRP; EC 1.11.1.7) were also able to conjugate GSH to CA and 4-CA. The peroxidase enzymes may generate thiyl free radicals of GSH that react with the alkyl double bond of CA and 4-CA resulting in the formation of a GSH conjugate.     

Reaction of the model thiol 2-mercaptoethanol and glutathione with methylvinylmaleimide,a Michael acceptor with extended conjugation

Many α,β-unsaturated compounds are sufficiently reactive to condense with nucleophiles under physiological conditions and are potentially deleterious to cellular processes. These compounds react with thiols by nucleophilic attack to give 1,4 addition products. We have examined the products formed from the reaction of the model thiols HSCH₂CH₂OH and glutathione with methylvinylmaleimide, a Michael acceptor with extended conjugation. Glutathione produced exclusively a 1,6 addition product with methylvinylmaleimide. HSCH₂CH₂OH also formed a 1,6 nucleophilic addition product, as well as a disubstituted product resulting from apparent further 1,4 addition to the 1,6 addition product. Two other novel products which resulted from addition to the maleimide ring and addition at the vinyl carbon proximal to the maleimide ring were observed.     

Detoxication of aliphatic epoxides by diol formation and glutathione conjugation

The Ames procedure with Salmonella typhimurium strain TA100 was used to follow the detoxication by rat liver fractions of two series of aliphatic epoxides. The epoxides employed were 3-chloro-, 3,3-dichloro- and 3,3,3-trichloropropylene oxides and also p-methoxyphenyl-, phenyl- and p-nitrophenylglycidyl ethers. In our procedure with preincubation of the epoxides with rat liver fractions prior to the Ames tests, there was more detoxication of both systems by glutathione conjugation (non-enzymatic and transferase promoted) than by the hydrolase pathways. Non-enzymatic reaction with glutathione was more pronounced for the chloro series than for the glycidyl ethers. An HPLC system was developed which was capable of quantitative measurements of the phenylglycidyl ethers together with their diol and glutathione conjugate products. A comparison of the HPLC and Ames test results indicates that the glutathione transferase reported to be present in Salmonella could be playing a role in detoxication by the Ames test. Diols were measured more readily by HPLC than by use of the Ames test in the microsomal fraction and were detected in the cytosol with the glycidyl ethers while they were not by the Ames procedure. However, all three epoxides were converted to a greater extent to their glutathione conjugates than to their diols. Thus, while literature references question the availability of the glutathione detoxication system for epoxides produced by membrane-bound enzymes, such detoxication would be of primary importance where direct-acting environmental epoxides come into contact with the cytosolic enzymes prior to possible reaction with bionucleophiles.     

Mutagenicity of tetrachloroethene in the ames test—metabolic activation by conjugation with glutathione

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S-(1,2,2-trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of γ-glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S-transferases in presence of GSH and rat kidney fractions resulted in a time-dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the β-lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by β-lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.     

Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines.

1. Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.     

Conjugations with glutathione. The enzymic conjugation of some chlorocyclohexenes

1. α-3,4,5,6-Tetrachlorocyclohex-1-ene and γ-2,3,4,5,6-pentachlorocyclohex-1-ene are conjugated with glutathione in vitro by a rat-liver enzyme that is probably glutathione S-aryltransferase. 2. Chlorocyclohexane and the α-, β-, γ- and δ-isomers of hexachlorocyclohexane were not substrates for rat-liver glutathione S-aryltransferase. 3. Glutathione-S-aryltransferase activity was present in tissue preparations of houseflies of insecticide-resistant and -susceptible strains. More activity was found in a dieldrin-resistant strain of houseflies fed on dieldrin than in either a dieldrin-resistant strain not fed on dieldrin or a control strain of dieldrin-susceptible houseflies. 4. Housefly soluble supernatant preparations converted S-(2-chloro-4-nitrophenyl)glutathione into the corresponding cysteine and mercapturic acid derivatives.     

An enzyme catalysing the conjugation of epoxides with glutathione

1. Liver supernatant preparations from rats and ferrets catalyse the conjugation of some epoxides with glutathione. The enzyme involved might be called `glutathione S-epoxidetransferase', as it is different from glutathione S-aryltransferase, the enzyme catalysing the conjugation of 1,2-dichloro-4-nitrobenzene, 4-nitro-pyridine N-oxide and other cyclic compounds with glutathione and from the enzyme catalysing the conjugation of iodomethane and glutathione. 2. The enzyme does not catalyse the reaction with cysteine. It is not inactivated by dialysis but is unstable at pH 5·0. 3. The role of the enzyme in metabolism of foreign compounds is discussed.     

Enzyme-catalysed conjugations of glutathione with unsaturated compounds

1. Rat-liver supernatant catalyses the reaction of diethyl maleate with glutathione. 2. Evidence is presented that the enzyme involved is different from the known glutathione-conjugating enzymes, glutathione S-alkyltransferase, S-aryltransferase and S-epoxidetransferase. 3. Rat-liver supernatant catalyses the reaction of a number of other αβ-unsaturated compounds, including aldehydes, ketones, lactones, nitriles and nitro compounds, with glutathione: separate enzymes may be responsible for these reactions.     

Binding and glutathione conjugation of porphyrinogens by plant glutathione transferases

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.     

Aminoacyl thiol esters and the origins of genetic specificity

Reasons for believing that primitive mechanisms of translation may have employed thiol esters of the amino acids rather than oxygen esters are summarized. It is suggested that coenzyme A (HSCoA), which fulfills the role of aminoacyl transfer in the synthesis of peptide antibiotics, is a primitive analogue of tRNA which performs a similar role in protein synthesis. HSCoA—an adenylic acid moiety containing phosphates esterified at the 3′ and 5′ positions and linked to a peptide-like structure terminating in a reactive thiol—possesses chemical features suggestive of both peptides and polynucleotides. Examination of the chemistry of HSCoA-like molecules shows that a rather similar compound can carry out a repeating intramolecular peptide synthesis in the absence of enzymes. Condensation of further nucleotides onto the adenylic acid moiety gives rise to parallel modes of peptide and oligonucleotide synthesis. A “self-improving” ability to select available amino acids is inherent in the proposed mechanism of peptide synthesis. The hypothesis plausibly explains the universal occurrence of a sulphur-containing amino acid at the N terminus of nascent proteins.     

Glutathione S-transferase-catalyzed conjugation of 9,10-epoxystearic acid with glutathione

The possible role of glutathione S-transferases (GST) in detoxification of fatty acid epoxides generated during lipid peroxidation has been evaluated. Present studies showed that cytosolic human glutathione S-transferases belonging to alpha, mu, and pi classes isolated from human liver and lung catalyzed the conjugation of glutathione and 9,10-epoxystearic acid. The product of enzymatic reaction, i.e., conjugate of GSH and epoxystearic acid, was isolated and characterized. The Michaelis constant (Km) values of the alpha, mu, and pi classes of GSTs for 9,10-epoxystearic acid were found to be 0.47, 0.32 and 0.80 mM, respectively, whereas the maximal velocity (V max) values for the alpha, mu, and pi classes of GSTs were found to be 142, 256, and 52 mol/min/mol, respectively. These results indicate that even though 9,10-epoxystearic acid is a substrate for all the three classes of GSTs, the mu class isozymes have maximum activity toward this substrate and may preferentially metabolize fatty acid epoxides more effectively as compared to the other classes of GSTs.     

Gramicidin channels in phospholipid bilayers with unsaturated acyl chains.

In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded beta(6,3)-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded pi pi(5,6)-helical dimers predominate (over single-stranded beta(6,3)-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded pi pi(5,6)-helical dimers in any of these different membrane environments. We conclude that the single-stranded beta(6,3)-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.     

The preparation of long chain-hydroxyacyl thiol esters: 3-hydroxyoctadecanoyl-CoA

Attempts to prepare 3-hydroxyoctadecanoyl-CoA by classical methods in fair yields failed, but it was synthesized in 30–35% yields by transesterification of 4-nitrophenyl 3-hydroxyoctadecanoate with the lithium salt of coenzyme A.