The kinetic mechanism of rat kidney gamma-glutamylcysteine synthetase.

A detailed kinetic investigation was made of the binding mechanism of gamma-glutamylcysteine synthetase purified from rat kidney. The results of initial rate and inhibition studies are consistent with a partially random mechanism in which ATP is the obligatory first substrate and both amino acids bind in a random order to the enzyme-ATP complex. Formation of the enzyme-substrate quaternary complex is necessary prior to release of products. This mechanism is consistent with previous binding studies with the enzyme and while it does not rule out participation of enzyme-bound gamma-glutamyl phosphate as an intermediate in catalysis, such an intermediate cannot be a discrete covalent complex.     

Amino acid sequence of rat kidney gamma-glutamylcysteine synthetase

gamma-Glutamylcysteine synthetase catalyzes the first step in the synthesis of glutathione. The enzyme isolated from rat kidney has two subunits (heavy, Mr 73,000; and light, Mr 27,700) which may be dissociated by treatment with dithiothreitol. The heavy subunit exhibits all of the catalytic activity of the isolated enzyme and also feedback inhibition by glutathione. The light subunit has no known function and may not be an integral part of the enzyme. cDNA clones encoding rat kidney gamma-glutamylcysteine synthetase were isolated from a lambda gt11 cDNA library by immunoscreening with antibody against the isolated enzyme and further screening with oligonucleotide probes derived from several peptides whose sequences were determined by the Edman method. The nucleotide sequence of the mRNA for the heavy subunit was deduced from the sequences of the cDNA of three such clones. The sequence, which codes for 637 residues (Mr 72,614), contains all four of the independently determined peptide sequences (approximately 100 residues). This amino acid sequence shows extremely low overall similarity to that of gamma-glutamylcysteine synthetase isolated from Escherichia coli.     

Isotope exchange at equilibrium studies with rat kidney gamma-glutamylcysteine synthetase

The kinetic binding mechanism of rat kidney gamma-glutamylcysteine synthetase has been evaluated by isotope exchange at equilibrium studies. The results are consistent with a Random BC mechanism as suggested previously (Yip, B. P., and Rudolph, F. B. (1976) J. Biol. Chem. 251, 3563-3568). The purification procedure for the enzyme was modified to remove contaminating adenylate kinase prior to isotope exchange studies.     

Knock down of gamma-glutamylcysteine synthetase in rat causes acetaminophen-induced hepatotoxicity

Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. Gamma-glutamylcysteine synthetase (gamma-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat gamma-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 x 10(11) pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development.     

Bovine lens gamma-glutamylcysteine synthetase. Inhibition by glutathione and adenine nucleotides

Steady-state kinetic analysis shows that glutathione binds reversibly to both Mg . enzyme and Mg . enzyme . L-glutamate forms of gamma-glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S-methyl glutathione are 16 mM and 0.5 mM, respectively. These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5'-diphosphate (5'ADP) is also an inhibitor and competes with both ATP and L-beta-chloroalanine for Mg . enzyme . L-glutamate and Mg . enzyme . L-glutamylphosphate, respectively. Under physiological conditions in the lens, 5' ADP competes effectively with L-cysteine for Mg . enzyme . L-glutamylphosphate, owing to the low concentration of L-cysteine, and less effectively with ATP for Mg . enzyme . L-glutamate, because of a high concentration of ATP.     

Bovine lenticular gamma-glutamylcysteine synthetase: reaction sequence.

The sequence of substrate addition and product release during the reaction catalyzed by gamma-glutamylcysteine synthetase was investigated with purified enzyme from bovine lens. Thermal inactivation and kinetic studies suggest that L-glutamate is the first substrate to bind to the enzyme. L-beta-Chloroalanine was used as the L-cysteine analogue. Utilizing substrate activation and product inhibition studies, the following reaction sequence was determined: L-glutamate binding. ATP binding, ADP release, L-beta-chloroalanine binding, followed by inorganic phosphate and then dipeptide release. The implications of this mechanism with regard to control of the enzyme in situ and its importance in glutathione synthesis are discussed.     

Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes

Rat hepatocytes cultured for 3 days in basal medium expressed low levels of cysteine dioxygenase (CDO) and high levels of gamma-glutamylcysteine synthetase (GCS). When the medium was supplemented with 2 mmol/l methionine or cysteine, CDO activity and CDO protein increased by >10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS) protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation, respectively. Supplementation with cysteine consistently yielded responses of greater magnitude than did supplementation with an equimolar amount of methionine. Addition of propargylglycine to inhibit cystathionine gamma-lyase activity and, hence, cysteine formation from methionine prevented the effects of methionine, but not those of cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine to inhibit GCS, and thus block glutathione synthesis from cysteine, did not alter the ability of methionine or cysteine to increase CDO. GSH concentration was not correlated with changes in either CDO or GCS-HS expression. The effectiveness of cysteine was equivalent to or greater than that of its precursors (S-adenosylmethionine, cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken together, these results suggest that cysteine itself is an important cellular signal for upregulation of CDO and downregulation of GCS.     

Regulation of gamma-glutamylcysteine synthetase expression in response to oxidative stress.

Glutathione (GSH) is synthesized by the activity of two ATP-requiring GSH synthesizing enzymes. Gamma-glutamylcysteine synthetase (gamma-GCS) is the rate limiting enzyme for the GSH synthesis. Gamma-GCS is a heterodimer of heavy, catalytic subunit and light, regulatory subunit and responsive to many stresses, such as heat shock, oxidative stress or cytokines. To know the regulation of the expression of gamma-GCS gene, in the present study, we show evidences that gamma-GCS heavy subunit is upregulated by oxidative stress by ionizing radiation and TNF-alpha mediated by nuclear factor-kappaB (NF-kappaB), and impairment of the expression of gamma-GCS by TNF-alpha in diabetic condition. Furthermore we describe the importance of GSH in the regulation of NF-kappaB subunits.     

Inactivation of human gamma-glutamylcysteine synthetase by cystamine. Demonstration and quantification of enzyme-ligand complexes.

Human erythrocyte gamma-glutamylcysteine synthetase is inactivated by the disulfide cystamine (2,2'-dithiobis-(ethylamine)) at pH 8.2 with a rate constant of 1020 min-1 mM-1. Magnesium ion and various combinations of substrates and products confer differing degrees of protection against cystamine inactivation, thus allowing the detection and quantification of certain enzyme-ligand interactions. By measuring inactivation rates as a function of ligand concentrations in incomplete reaction mixtures, we have obtained evidence for the following complexes: enzyme . Mg2+; enzyme . Mg2+ . MgATP2-; enzyme . Mg2+ . L-glutamate; enzyme . Mg2+ . MgATP2- . L-glutamate; enzyme . Mg2+ . L-gamma-glutamyl-L-alpha-aminobutyrate. The data also imply the existence of enzyme . (Mg2+)2 . MgATP2- . L-glutamate and several enzyme forms resulting from the weak binding to L-alpha-aminobutyrate. The methods used permit the calculation of cystamine inactivation rates for most of these enzyme forms and also give values for the equilibrium constants describing their formation.     

Inactivation of gamma-glutamylcysteine synthetase, but not of glutamine synthetase, by S-sulfocysteine and S-sulfohomocysteine

The reactions catalyzed by gamma-glutamylcysteine synthetase and glutamine synthetase are thought to proceed via enzyme-bound gamma-glutamyl phosphate intermediates. We investigated the possibility that S-sulfocysteine and S-sulfohomocysteine might act as analogs of gamma-glutamyl phosphate or of the associated putative tetrahedral intermediates. The D- and L-enantiomers of S-sulfocysteine and S-sulfohomocysteine were found to rapidly inactivate rat kidney gamma-glutamylcysteine synthetase but to be reversible inhibitors of sheep brain glutamine synthetase. Inactivation of gamma-glutamylcysteine synthetase does not require ATP and is associated with noncovalent binding of close to 1 mol of inactivator/mol of enzyme. The findings indicate that the S-sulfo amino acids are transition-state analogs, and that binding of S-sulfo amino acid to the enzyme induces formation of a very stable enzyme-inactivator complex. The data suggest that stabilization of the enzyme-inactivator complex results from interactions involving the sulfenyl sulfur atom of the S-sulfo amino acid and the active site thiol group of the enzyme.     

Melatonin induces gamma-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells.

In the present study, we show that melatonin induces the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione (GSH) synthesis, in ECV304 human vascular endothelial cells. One micromolar melatonin induced the expression of gamma-GCS mRNA followed by an increase in the concentration of GSH with a peak at 24 h. An electrophoretic mobility shift assay showed that melatonin stimulates the DNA-binding activity of activator protein-1 (AP-1) as well as retinoid Z receptor/retinoid receptor-related orphan receptor alpha (RZR/RORalpha). ECV304 cells transiently transfected with a plasmid containing the gamma-GCS promoter-luciferase construct showed increased luciferase activity when treated with melatonin. The melatonin-dependent luciferase activity was found in the gamma-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 was repressed in the promoter containing RZR/RORalpha site. In addition, cell cycle analysis showed that melatonin increases the number of cells in the G0/G1 phase; however, treatment of the cells with buthionine sulfoximine, a specific inhibitor of gamma-GCS, abolished the effect of melatonin on the cell cycle, suggesting induction of cell arrest by melatonin requires GSH. As conclusion, induction of GSH synthesis by melatonin protects cells against oxidative stress and regulates cell proliferation.     

Induction of gamma-glutamylcysteine synthetase by prostaglandin A2 in L-1210 cells

Effects of prostaglandin A2 (PGA2) on glutathione (GSH) status in L-1210 cells were examined. When the cells were cultured in the presence of PGA2, a persistent rise of cellular GSH concentration was observed 6 h after the addition of PGA2. This stimulatory effect of PGA2 was abolished if the cells were pretreated with an enzyme inhibitor of GSH synthesis, buthionine sulfoximine. Subsequent study with cell free extract of cultured L-1210 has revealed that PGA2 stimulated the biosynthesis of gamma-glutamylcysteine synthetase (EC 6.3.2.2). Actinomycin D inhibited this stimulatory effect of PGA2 on cultured cells. The optimal pH, Km value for glutamic acid and sensitivity to inhibitors of gamma-glutamylcysteine synthetase from PGA2 treated and nontreated cells were virtually the same. Thus, our findings suggest that PGA2 induced gamma-glutamylcysteine synthetase in cultured L-1210 cells which is responsible for the elevated level of GSH in these cells.     

Inducers of gamma-glutamylcysteine synthetase and their effects on glutathione synthetase expression

Synthesis of GSH occurs via two enzymatic steps, the first is catalyzed by gamma-glutamylcysteine synthetase (GCS) and the second is catalyzed by GSH synthetase (GS). A heavy (HS) and light subunit (LS) make up GCS; regulation of both subunits have been well characterized, whereas regulation of GS is largely unknown. In this study, we examined the effects of treatments known to influence the gene expression of GCS subunits on GS expression. Insulin and hydrocortisone treatment of rat hepatocytes or ethanol-feeding of rats for 9 weeks, which increased the expression of GCS-HS only, had no influence on GS expression. However, two-thirds partial hepatectomy in rats which increased the expression of GCS-HS only, also increased GS expression. Treatment of hepatocytes or rats with diethyl maleate, buthionine sulfoximine, tert-butylhydroquinone, or thioacetamide, which increased the expression of both GCS subunits, increased the expression of GS. The GSH synthesis capacity increased 50-100% by treatments that increased only the GCS-HS expression, whereas it increased 161-200% by treatments that increased both GCS-HS and GS expression. Thioacetamide treatment of Chang cells increased cell GSH and GS expression by 50%, but had minimal influence on GCS subunits. Thus, GS induction can further increase the cell's GSH synthetic capacity and in some cells may be as important as GCS in determining the rate of GSH synthesis.     

Glutathione synthesis in Streptococcus agalactiae. One protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities

Gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the gamma-GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae gamma-GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L-gamma-glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L-gamma-glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as gamma-GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of gamma-GCS-GS (total M(r) 85,000) show 32% identity and 43% similarity with E. coli gamma-GCS (M(r) 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of gamma-GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.     

Age-associated decline in gamma-glutamylcysteine synthetase gene expression in rats

Although glutathione (GSH) concentration has been reported to diminish with age, the mechanism underlying such age-associated decline in the GSH content is not well understood. In this study, we compared the gene expression of both subunits of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, in young, adult, and old Fisher 344 rats. It was found that GCS activity was significantly decreased with increased age in liver, kidney, lung, and red blood cells (RBC). Parallel with the decreased enzyme activity, the protein and mRNA contents of both GCS subunits also changed inversely with age in liver, kidney, and lung, implying a decreased GCS gene expression during aging. Such a reduced GCS gene expression was accompanied by a decline in total GSH content without any change in cysteine concentration. Furthermore, the decreased GCS gene expression in old rats was not associated with a decline in the plasma insulin or cortisol level. This study showed, for the first time, that the expression of both GCS subunit genes was decreased in some organs of old rats, which would result in a reduced rate of GSH biosynthesis. Such decline in GSH synthetic capacity may underlie the observed decrease in GSH content during aging.