Step In Glutathione Biosynthesis Research Articles

Glutathione overproduction mediates lymphoma initiating cells survival and has a sex-dependent effect on lymphomagenesis

Lymphoid tumor patients often exhibit resistance to standard therapies or experience relapse post-remission. Relapse is driven by Tumor Initiating Cells (TICs), a subset of tumor cells capable of regrowing the tumor and highly resistant to therapy. Growing cells in 3D gels is a method to discern tumorigenic cells because it strongly correlates with tumorigenicity. The finding that TICs, rather than differentiated tumor cells, grow in 3D gels offers a unique opportunity to unveil TIC-specific signaling pathways and therapeutic targets common to various cancer types. Here, we show that culturing lymphoid cells in 3D gels triggers reactive oxygen species (ROS) production, leading to non-tumor lymphoid cell death while enabling the survival and proliferation of a subset of lymphoma/leukemia cells, TICs or TIC-like cells. Treatment with the antioxidant N-acetylcysteine inhibits this lethality and promotes the growth of primary non-tumor lymphoid cells in 3D gels. A subset of lymphoma cells, characterized by an increased abundance of the antioxidant glutathione, escape ROS-induced lethality, a response not seen in non-tumor cells. Reducing glutathione production in lymphoma cells, either through pharmacological inhibition of glutamate cysteine ligase (GCL), the enzyme catalyzing the rate-limiting step in glutathione biosynthesis, or via knockdown of GCLC, the GCL catalytic subunit, sharply decreased cell growth in 3D gels and xenografts. Tumor cells from B-cell lymphoma/leukemia patients and λ-MYC mice, a B-cell lymphoma mouse model, overproduce glutathione. Importantly, pharmacological GCL inhibition hindered lymphoma growth in female λ-MYC mice, suggesting that this treatment holds promise as a therapeutic strategy for female lymphoma/leukemia patients.

Genetic variation at the catalytic subunit of glutamate cysteine ligase contributes to the susceptibility to sporadic colorectal cancer: a pilot study.

Glutathione is a tripeptide detoxifying a variety of exogenous and endogenous free radicals and carcinogens, and a deficiency of glutathione is associated with an increased host susceptibility to oxidative stress, a pathological condition implicated in the development and progression of cancer. The catalytic subunit of glutamate-cysteine ligase (GCLC) is an enzyme responsible for the initial and rate-limiting step of glutathione biosynthesis. The aim of this pilot study was to investigate whether genetic variation at the GCLC gene contributes to the risk of colorectal cancer (CRC). DNA samples from 681 unrelated Russian individuals (283 patients with CRC and 398 age- and sex-matched healthy controls) were genotyped for six common functional SNPs of the GCLC gene (SNPs) such as rs12524494, rs17883901, rs606548, rs636933, rs648595 and rs761142 of the GCLC gene using the MassARRAY-4 system. We found that genotype rs606548-C/T is significantly associated with increased risk of CRC regardless of sex and age (OR 2.24; 95% CI 1.24-4.03; P = 0.007, FDR = 0.04). Moreover, ten GCLC genotype combinations showed association with the risk of CRC (P < 0.05). Functional SNP annotation enabled establishing the CRC-associated polymorphisms are associated with a decreased GCLC expression that may be attributed to epigenetic effects of histone modifications operating in a colon-specific manner. The present study was the first to show that genetic variation at the catalytic subunit of glutamate-cysteine ligase may contribute to the risk of colorectal cancer risk. However, further genetic association studies with a larger sample size are required to substantiate the role of GCLC gene polymorphisms in the development of sporadic colorectal cancer.

A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair.

The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing.

Selenite Protection of Tellurite Toxicity Toward Escherichia coli.

In this work the influence of selenite on metal resistance in Escherichia coli was examined. Both synergistic and antagonistic resistance and toxicities were found upon co exposure with selenite. In wild type cells co-exposure to selenite had little effect on arsenic resistance, decreased resistance to cadmium and mercury but led to a dramatically increased resistance to tellurite of 32-fold. Due to the potential importance of thiol chemistry in metal biochemistry, deletion strains in γ-glutamylcysteine synthetase (key step in glutathione biosynthesis, encoded by gshA), thioredoxin (trxA), glutaredoxin (grxA), glutathione oxidoreductase (gor), and the periplasmic glutathione transporter (cydD) were also evaluated for resistance to various metals in the presence of selenite. The protective effect of selenite on tellurite toxicity was seen in several of the mutants and was pronounced in the gshA mutant were resistance to tellurite was increased up to 1000-fold relative to growth in the absence of selenite. Thiol oxidation studies revealed a faster rate of loss of reduced thiol content in the cell with selenite than with tellurite, indicating differential thiol reactivity. Selenite addition resulted in reactive oxygen species (ROS) production equivalent to levels associated with H2O2 addition. Tellurite addition resulted in considerably lower ROS generation while vanadate and chromate treatment did not increase ROS production above that of background. This work shows increased resistance toward most oxyanions in mutants of thiol redox suggesting that metalloid reaction with thiol components such as glutathione actually enhances toxicity of some metalloids.

Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation

Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalysed by γ-GCS (γ-glutamylcysteine synthetase). The crystal structure of Escherichia coli γ-GCS has revealed the presence of a disulfide bond. As the disulfide-bonding cysteine residues Cys372 and Cys395 are not well conserved among γ-GCS enzymes in this lineage, we have initiated a biochemical genetic strategy to investigate the functional importance of these and other cysteine residues. In a cysteine-free γ-GCS that was non-functional, suppressor analysis yielded combinations of cysteine and aromatic residues at the position of the disulfide bond, and one mutant that lacked any cysteine residues. Kinetic analysis of the wild-type and mutant enzymes revealed that the disulfide bond was not involved in determining the affinity of the enzyme towards its substrate, but had an important role in determining the stability of the protein, and its catalytic efficiency. We show that in vivo the γ-GCS enzyme can also exist in a reduced form and that the mutants lacking the disulfide bond show a decreased half-life. These results demonstrate a novel means of regulation of γ-GCS by the redox environment that works by an alteration in its stability.

P48 Cystathionine gamma lyase mediates its anti-inflammatory and cytoprotective functions through protein sulfhydration

Cystathionine gamma lyase (CSE) also known as γ-cystathionase, is a key enzyme in the transsulfuration pathway leading to the synthesis of glutathione, the major antioxidant in the cell. CSE converts cystathionine to cysteine, the availability of which is the rate limiting step in glutathione biosynthesis. In addition, CSE utilizes cysteine to generate hydrogen sulfide (H2S), which is one of the three gasotransmitters, the other two being nitric oxide and carbon monoxide. H2S plays an important role in diverse signaling pathways. One of the mechanisms by which H2S signals is via the formation of a persulfide (-SSH) bond at reactive Cys residues, a process designated sulfhydration. Using a modified biotin switch assay, we have shown that sulfhydration of target proteins such as cyclooxygenase 2 (COX2) and nuclear factor κB (NF-κB) modulates their activity thereby regulating prosurvival and inflammatory responses. Using CSE knockout mice we show that H2S functions as an anti-inflammatory and antioxidant molecule in vivo. Depletion of CSE led to a decrease in levels of glutathione and increase in oxidative stress as revealed by protein carbonylation, lipid peroxidation and DNA damage detection assays. Primary hepatocytes and mouse embryo fibroblasts isolated from CSE knockout mice had higher levels of reactive oxygen species and exhibited a higher susceptibility to oxidants such as hydrogen peroxide. Our studies reveal an important role for CSE in the maintenance of redox homeostasis in the cell.

Polymorphism in glutamate cysteine ligase catalytic subunit (GCLC) is associated with sulfamethoxazole-induced hypersensitivity in HIV/AIDS patients

BackgroundSulfamethoxazole (SMX) is a commonly used antibiotic for prevention of infectious diseases associated with HIV/AIDS and immune-compromised states. SMX-induced hypersensitivity is an idiosyncratic cutaneous drug reaction with genetic components. Here, we tested association of candidate genes involved in SMX bioactivation and antioxidant defense with SMX-induced hypersensitivity.ResultsSeventy seven single nucleotide polymorphisms (SNPs) from 14 candidate genes were genotyped and assessed for association with SMX-induced hypersensitivity, in a cohort of 171 HIV/AIDS patients. SNP rs761142 T > G, in glutamate cysteine ligase catalytic subunit (GCLC), was significantly associated with SMX-induced hypersensitivity, with an adjusted p value of 0.045. This result was replicated in a second cohort of 249 patients (p = 0.025). In the combined cohort, heterozygous and homozygous carriers of the minor G allele were at increased risk of developing hypersensitivity (GT vs TT, odds ratio = 2.2, 95% CL 1.4-3.7, p = 0.0014; GG vs TT, odds ratio = 3.3, 95% CL 1.6 – 6.8, p = 0.0010). Each minor allele copy increased risk of developing hypersensitivity 1.9 fold (95% CL 1.4 – 2.6, p = 0.00012). Moreover, in 91 human livers and 84 B-lymphocytes samples, SNP rs761142 homozygous G allele carriers expressed significantly less GCLC mRNA than homozygous TT carriers (p < 0.05).Conclusionsrs761142 in GCLC was found to be associated with reduced GCLC mRNA expression and with SMX-induced hypersensitivity in HIV/AIDS patients. Catalyzing a critical step in glutathione biosynthesis, GCLC may play a broad role in idiosyncratic drug reactions.

Management of pediatric epilepsies

S cells are pluripotential cells that have the ability to differentiate into cell lineages that are determined by trophic factors and other biological cues. There is growing evidence in neonatal medicine which suggests understanding stem cell physiology may offer a unique pathway leading to decreased pathology. Researchers have proposed bone marrow-derived stem cell use in the treatment of chronic lung disease (Alphonse et al., 2012). In a similar fashion, mesenchymal stem cells also protected against lung disease (Tian et al., 2008). In these studies stem cells show a reparative mechanism that is currently not well described. Mesenchymal stem cells have also been shown to decrease cellular insults in an animal model of necrotizing enterocolitis (Tayman et al 20011). Stem cells have been administered intranasally and shown to have a protective effect on brain injury (van Velthaven 2010). Our laboratory has uncovered a potentially beneficial pathway that involves the biosynthesis of glutathione in stem cells. Most neonatal diseases involve some form of oxidative stress. Glutathione, a tripeptide, is the endogenous tool used to combat oxidative stress. This pathway involves the regulation of the cystine glutamate exchanger (System Xc) which controls the most critical step in glutathione biosynthesis. This pathway is one of the mechanisms that stem cells use to exert its’ protective effect.

Structural Basis for Feedback and Pharmacological Inhibition of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A; R = 19.9%, R(free) = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site and disrupts the previously observed Mg(2+) coordination in the ATP-binding site. l-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 A; R = 18.1%, R(free) = 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.

Dietary Selenium (Se) and Copper (Cu) Interact to Affect Homocysteine Metabolism in Rats

Previously, we reported that both Se deficiency and Cu deficiency decreased plasma homocysteine (pHcys) and increased plasma glutathione (pGSH) in rats. We also showed that the catalytic subunit of glutamate-cysteine ligase (Gclc), which catalyzes the rate-limiting step in glutathione biosynthesis, was upregulated in Se and Cu deficiencies. We suggested that in both deficiencies, Hcys was being shunted through the trans-sulfuration pathway as a result of this up-regulation. Because both Se and Cu deficiencies have similar effects, we hypothesized that a combined deficiency would exacerbate the decrease in pHcys and the increase in pGSH by further up-regulating Gclc. In a 2 x 2 experiment, male weanling Sprague-Dawley rats (n = 8-20/group) were fed an amino-acid-based diet containing either 0 or 0.2 microg Se (as selenite)/g and <1 or 6 microg Cu (as Cu carbonate)/g for 5 weeks. Our findings show that a combined deficiency of both Se and Cu results in lower pHcys and significantly elevated pGSH. However, the up-regulation of liver Gclc alone cannot explain why rats fed with the doubly deficient diet have the lowest pHcys and the highest pGSH.

Expression, purification and characterization of glutamate cysteine ligase from Saccharomyces cerevisiae

Glutathione is a critical cellular reductant and its homeostasis is required for maintenance of intracellular redox balance. glutamate‐cysteine ligase (GCL) catalyses the ATP‐dependent peptide bond formation between gamma‐carboxylate of glutamate and cysteine, the first step of glutathione biosynthesis. Mammalian GCL are heterodimers consisting of a catalytic subunit and a modifier subunit. The catalytic subunit catalyzes formation of glutamylcysteine and is inhibited by GSH, whereas the modifier subunit enhances the GCL affinity for L‐glutamate and decreases the inhibition by GSH. An examination of the S. cerevisiae genome revealed a single gene with sequence homology to the catalytic GCL subunit. The S. cerevisiae GCL catalytic subunit was cloned, expressed in E. coli and purified to homogeneity. Size exclusion chromatography and DLS techniques were used to examine the oligomeric state of GCL catalytic subunit. The kinetic activity of GCL catalytic subunit was measured using a coupled assay and apparent Km and Vmax values were determined. Kinetic constants were comparable to those reported for other eukaryotic GCL, and inhibition studies indicate that the enzyme is susceptible to competitive inhibition by the ultimate product of the pathway, glutathione. A proteomic method is currently being developed to identify the corresponding modifier subunit of S. cerevisiae GCL.

Dietary selenium (Se) and copper (Cu) interact to affect homocysteine metabolism in rats

Previously we reported that both Se deficiency (SeD) and Cu deficiency (CuD) decreased plasma homocysteine (pHcys) and increased plasma glutathione (pGSH) in rats. We also showed that the catalytic subunit of glutamate‐cysteine ligase (Gclc), which catalyzes the rate limiting step in glutathione biosynthesis, was upregulated in SeD as well as in CuD. We suggested that in both deficiencies, that Hcys was being shunted through the transsulfuration pathway as a result of this upregulation. Because both SeD and CuD have similar effects, we hypothesized that a combined deficiency would exacerbate the decrease in pHcys and the increase in pGSH by further upregulating Gclc. In a 2 × 2 experiment, male weanling Sprague‐Dawley rats (N=8–10/group) were fed an amino acid‐based diet containing either 0 or 0.2 μg Se (as selenite)/g and &lt;1 or 6 μg Cu (as Cu carbonate)/g for 5 wk. Values in the table are means±SD. Both SeD and CuD resulted in decreased pHcys; however, CuD in rats fed 0.2 μg Se/g resulted in a greater decrease in pHcys than did CuD in the SeD rats. SeD and CuD resulted in increased pGSH and the combined deficiencies further exacerbated the increase. Plasma cysteine (pCys) was decreased by SeD and increased by CuD.The findings show that a combined deficiency of both Se and Cu results in lower pHcys and significantly elevated pGSH most likely as a result of increased GSH biosynthesis subsequent to upregulation of Gclc.

Copper Deficiency Decreases Plasma Homocysteine in Rats

The purpose of this study was to determine the effects of copper deficiency on key aspects of homocysteine metabolism that involve methionine recycling and transsulfuration. Male weanling Sprague-Dawley rats were fed AIN-93G-based diets containing <1 or ∼6 mg Cu/kg. After 6 wk (Expt. 1) and 4 wk (Expt. 2) we found that plasma homocysteine was significantly decreased, and plasma glutathione significantly increased, in rats fed the low-Cu diet. Real-time RT-PCR was used to determine the expression of the subunits of glutamate-cysteine ligase (Gcl) in liver that catalyzes the rate-limiting step in glutathione biosynthesis. The expression of Gclc, the catalytic subunit of Gcl, was upregulated by Cu deficiency; Gclm, the modifier subunit, was not affected. Hepatic betaine-homocysteine methyltransferase (Bhmt), which catalyzes one of the two ways that homocysteine can be remethylated to methionine, was downregulated by Cu deficiency. Because Cu deficiency results in upregulation of Gclc and an increase in the biosynthesis of glutathione, it is plausible that the net flux of homocysteine through the transsulfuration pathway is increased. Furthermore, if Bhmt is downregulated, less homocysteine is available for remethylation (methionine recycling) and more is then available to irreversibly enter the transsulfuration pathway where it is lost. The net effect of increased Gclc and decreased Bhmt would be a decrease in homocysteine as a result of Cu deficiency.

Dietary Selenium Affects Homocysteine Metabolism Differently in Fisher-344 Rats and CD-1 Mice ,4

In our previous work with rats, plasma and tissue homocysteine concentrations were decreased by selenium deprivation. The purpose of this study was to follow up and expand on that work by determining the effects of selenium status (deficient, adequate, and supranutritional) on several aspects of homocysteine metabolism involving methionine recycling and transsulfuration. A 2nd objective was to determine whether there are differences in how selenium status affects homocysteine metabolism in rats and mice. Male weanling Fischer-344 rats and male weanling CD-1 mice were fed diets containing 0, 0.2, or 2.0 μg selenium (as sodium selenite)/g for 72 d or 60 d, respectively. Plasma homocysteine and cysteine were significantly decreased by feeding rats or mice the selenium-deficient diet compared with feeding adequate or supranutritional selenium. On the other hand, plasma glutathione was increased by selenium deficiency only in rats. Also, the specific activities of liver betaine homocysteine methyltransferase and glycine N-methyltransferase were decreased by selenium deficiency in rats, but were unaffected by selenium status in mice. Real-time RT-PCR was used to determine the expression of the subunits of glutamate-cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis. The expression of Gclc, the catalytic subunit of glutamate-cysteine ligase, was upregulated by selenium deprivation in both rat and mouse liver. Gclm, the modifier subunit of glutamate-cysteine ligase, was downregulated in rats fed 2 μg Se/g compared with rats fed adequate or deficient selenium. Based on these findings, it is evident that selenium deficiency has different outcomes in mice and rats. These variables are all related to methionine/methyl metabolism. Although only one strain of rat was compared with one strain of mouse, this work suggests that differences between species may prove vital in determining which animal model is used in studies of selenium deficiency or in studies that are designed to ascertain chemopreventive mechanisms of selenium.

Redox‐Regulation of Glutathione Synthesis in Plants

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamate-cysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes; however, kinetic analysis shows that AtGCL is functionally similar to the enzyme from other organisms. Buthionine sulfoximine and cystamine inactivate AtGCL, but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. Global fitting analysis of initial velocity data indicates that a random ter-reactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consist of a catalytic subunit and a regulatory subunit, AtGCL functions as a monomeric protein. In response to changes in redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. Because reducing agents mediate this change, an intramolecular disulfide bond is likely involved. Thus, regulation of AtGCL does not require association with a regulatory subunit, as in the mammalian system, for activation. This model for controlling AtGCL activity explains observations that oxidative stimulus induces glutathione synthesis without increased gene expression in plants. Similar redox signal-induced conformational switching by disulfide formation occurs in other proteins, including Yap1, thioredoxins, and OxyR.

Oxidant-specific Folding of Yap1p Regulates Both Transcriptional Activation and Nuclear Localization

The yeast transcriptional regulator Yap1p is a key determinant in oxidative stress resistance. This protein is found in the cytoplasm under non-stressed conditions but rapidly accumulates in the nucleus following oxidant exposure. There it activates transcription of genes encoding antioxidants that return the redox balance of the cell to an acceptable range. Yap1p localization to the nucleus requires the oxidant-specific formation of disulfide bonds in the N-terminal cysteine-rich domain (N-CRD) and/or the C-terminal cysteine-rich domain (C-CRD). H(2)O(2) exposure triggers the formation of two interdomain disulfide bonds between the N-and C-CRDs. This dually disulfide-bonded structure has been argued to mask the nuclear export signal in the C-CRD that would otherwise prevent Yap1p nuclear accumulation. The C-CRD is required for wild-type H(2)O(2) tolerance but dispensable for resistance to diamide. The Saccharomyces cerevisiae TRX2 gene, encoding a thioredoxin protein, cannot be induced by H(2)O(2) in the presence of various mutant forms of Yap1p lacking the normally functioning C-CRD. In this work, we demonstrate that the proper folding of Yap1p in the presence of H(2)O(2) is required for recruitment of the mediator component Rox3p to the TRX2 promoter in addition to the nuclear accumulation of Yap1p during stress by this oxidant. These data demonstrate that the dually disulfide-bonded Yap1p N- and C-CRDs form a bifunctional protein domain controlling both nuclear localization and transcriptional activation.

Arabidopsis thaliana Glutamate-Cysteine Ligase: FUNCTIONAL PROPERTIES, KINETIC MECHANISM, AND REGULATION OF ACTIVITY

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of gamma-glutamylcysteine from glutamate (Km = 9.1 mm) and cysteine (Km = 2.7 mm). Glutathione feedback inhibits AtGCL (Ki approximately 1.0 mm). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random terreactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress.

Large Conformational Changes in the Catalytic Cycle of Glutathione Synthase

Glutathione synthase catalyzes the final ATP-dependent step in glutathione biosynthesis, the formation of glutathione from γ-glutamylcysteine and glycine. We have determined structures of yeast glutathione synthase in two forms: unbound (2.3 Å resolution) and bound to its substrate γ-glutamylcysteine, the ATP analog AMP-PNP, and two magnesium ions (1.8 Å resolution). These structures reveal that upon substrate binding, large domain motions convert the enzyme from an open unliganded form to a closed conformation in which protein domains completely surround the substrate in the active site.

Buthionine sulfoximine embryotoxicity is associated with prolonged AP‐1 activation

Many teratogens induce oxidative stress, altering redox status and redox signaling; this has led to the suggestion that developmental toxicants act by disturbing redox status. The goal of these studies was to determine the consequences of altering glutathione homeostasis during organogenesis on embryo development, total DNA methylation, and activator protein-1 (AP-1) DNA binding activity and gene expression. Gestational day 10.5 rat embryos were cultured in vitro for up to 44 hour in the presence of L-buthionine-S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamyl-cysteine synthetase, the rate limiting step in glutathione biosynthesis. Effects of BSO on total, oxidized and reduced glutathione, embryo development, DNA methylation, AP-1 DNA binding activity and gene expression were investigated. Significant depletion of glutathione by BSO was first noted at 6 hr in the embryo and at 3 hr in the yolk sac; total glutathione in the conceptus was depleted to the same extent after treatment with either 0.1 or 1.0 mM BSO. Exposure to 0.1 mM BSO did not cause a significant increase in embryotoxicity, although some impairment of growth and development was observed. In contrast, exposure to 1.0 mM BSO severely inhibited growth and development, significantly increasing the incidence of swollen hindbrains and of blebs in the forebrain, limb and maxillary regions. No significant treatment-related differences in total DNA methylation were observed. Interestingly, AP-1 DNA binding activity was similar in control and 0.1 mM BSO-treated conceptuses; however, exposure to 1.0 mM BSO increased AP-1 DNA binding at 6, 24, and 44 hr. The expression of several AP-1 family genes and of gamma-glutamylcysteine synthetase was induced in embryos cultured with 1.0 mM BSO. Exposure of embryos in vitro to BSO at a concentration that was embryotoxic induced prolonged AP-1 DNA binding activity and altered gene expression. These data suggest that AP-1 induction may serve as a biomarker of embryo stress.

Escherichia coli γ-Glutamylcysteine Synthetase: TWO ACTIVE SITE METAL IONS AFFECT SUBSTRATE AND INHIBITOR BINDING

Gamma-glutamylcysteine synthetase (gamma-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes. Although all eukaryotic gamma-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial gamma-GCS is resistant to BSO. We have confirmed the latter finding with Escherichia coli gamma-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms. We also find that substitution of Mn2+ for Mg2+ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation. Similarly, the specificity of E. coli gamma-GCS for its amino acid substrates is broadened in the presence of Mn2+, and the rate of reaction for some very poor substrates is improved. These results suggest that divalent metal ions have a role in amino acid binding to E. coli gamma-GCS. Electron paramagnetic resonance (EPR) studies carried out with Mn2+ show that E. coli gamma-GCS binds two divalent metal ions; Kd values for Mn2+ are 1.1 microm and 82 microm, respectively. Binding of l-glutamate or l-BSO to the two Mn2+/gamma-GCS species produces additional upfield and downfield X-band EPR hyperfine lines at 45 G intervals, a result indicating that the two Mn2+ are spin-coupled and thus apparently separated by 5 A or less in the active site. Additional EPR studies in which Cu2+ replaced Mg2+ or Mn2+ suggest that Cu2+ is bound by one N and three O ligands in the gamma-GCS active site. The results are discussed in the context of the catalytic mechanism of gamma-GCS and its relationship to the more fully characterized glutamine synthetase reaction.