Psychrophilic superoxide dismutase from Pseudoalteromonas haloplanktis: biochemical characterization and identification of a highly reactive cysteine residue

A psychrophilic superoxide dismutase (SOD) has been characterized from the Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph). PhSOD is a homodimeric iron-containing enzyme and displays a high specific activity, even at low temperature. The enzyme is inhibited by sodium azide and inactivated by hydrogen peroxide; it is also very sensitive to peroxynitrite, a physiological inactivator of the human mitochondrial Mn-SOD. Even though PhSOD is isolated from a cold-adapted micro-organism, its heat stability is well above the maximum growth temperature of P. haloplanktis, a feature common to other Fe- and Mn-SODs. The primary structure of PhSOD was determined by a combination of mass spectrometry and automated Edman degradation. The polypeptide chain is made of 192 amino acid residues, corresponding to a molecular mass of 21251 Da. The alignment with other Fe- and Mn-SODs showed a high amino acid identity with Fe-SOD from Vibrio cholerae (79%) and Escherichia coli (70%). A significant similarity is also shared with human mitochondrial Mn-SOD. PhSOD has the unique and highly reactive Cys57 residue, located in a variable region of the protein. The three-dimensional model of the PhSOD monomer indicates that Cys57 is included in a region, whose structural organization apparently discriminates between dimeric and tetrameric SODs. This residue forms a disulfide adduct with beta-mercaptoethanol, when this reducing agent is added in the purification procedure. The reactivity of Cys57 leads also to the formation of a disulfide bridge between two PhSOD subunits in specific denaturing conditions. The possible modification of Cys57 by physiological thiols, eventually regulating the PhSOD functioning, is discussed.     

S-nitrosylation and S-glutathionylation of GAPDH: Similarities,differences, and relationships

The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation.Incubation of HEK 293 T cells in the presence of H₂O₂ or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H₂O₂ or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.     

Redox regulation of a soybean tyrosine-specific protein phosphatase

Plant protein tyrosine phosphatases (PTPs) are important in regulating cellular responses to redox change through their reversible inactivation under oxidative conditions. Studies on the soybean (Glycine max) GmPTP have shown that, compared with its mammalian counterparts, the plant enzyme is relatively insensitive to inactivation by H2O2 but hypersensitive (k(inact) = 559 M(-1) s(-1)) to S-glutathionylation (thiolation) promoted by the presence of oxidized glutathione (GSSG). Through a combination of chemical and mutational modification studies, three of the seven cysteine residues of GmPTP have been identified by mass spectrometry as being able to inactivate the enzyme when thiolated by GSSG or alkylated with iodoacetamide. Conserved Cys 266 was shown to be essential for catalysis but surprisingly resistant to S-modification, whereas the regulatory Cys 78 and Cys 176 were readily thiolated and/or alkylated. Mutagenesis of these cysteines showed that all three residues were in proximity of each other, regulating each's reactivity to S-modifying agents. Through a combination of protein modification and kinetic experiments, we conclude that the inactivation of GmPTP by GSSG is regulated at two levels. Cys 176 appears to be required to promote the formation of the reduced form of Cys 266, which is otherwise unreactive. When thiolated, Cys 176 immediately inactivates the enzyme, and this is followed by the thiolation of Cys 78, which undergoes a slow disulfide exchange with Cys 266 giving rise to a Cys 78-Cys 266 disulfide. We speculate that this two-tiered protection is required for regulation of GmPTP under highly oxidizing conditions.     

Molecular basis and structural insight of vascular K(ATP) channel gating by S-glutathionylation

The vascular ATP-sensitive K(+) (K(ATP)) channel is targeted by a variety of vasoactive substances, playing an important role in vascular tone regulation. Our recent studies indicate that the vascular K(ATP) channel is inhibited in oxidative stress via S-glutathionylation. Here we show evidence for the molecular basis of the S-glutathionylation and its structural impact on channel gating. By comparing the oxidant responses of the Kir6.1/SUR2B channel with the Kir6.2/SUR2B channel, we found that the Kir6.1 subunit was responsible for oxidant sensitivity. Oxidant screening of Kir6.1-Kir6.2 chimeras demonstrated that the N terminus and transmembrane domains of Kir6.1 were crucial. Systematic mutational analysis revealed three cysteine residues in these domains: Cys(43), Cys(120), and Cys(176). Among them, Cys(176) was prominent, contributing to >80% of the oxidant sensitivity. The Kir6.1-C176A/SUR2B mutant channel, however, remained sensitive to both channel opener and inhibitor, which indicated that Cys(176) is not a general gating site in Kir6.1, in contrast to its counterpart (Cys(166)) in Kir6.2. A protein pull-down assay with biotinylated glutathione ethyl ester showed that mutation of Cys(176) impaired oxidant-induced incorporation of glutathione (GSH) into the Kir6.1 subunit. In contrast to Cys(176), Cys(43) had only a modest contribution to S-glutathionylation, and Cys(120) was modulated by extracellular oxidants but not intracellular GSSG. Simulation modeling of Kir6.1 S-glutathionylation suggested that after incorporation to residue 176, the GSH moiety occupied a space between the slide helix and two transmembrane helices. This prevented the inner transmembrane helix from undergoing conformational changes necessary for channel gating, retaining the channel in its closed state.     

Properties of Cys21-mutated muscle acylphosphatases

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed inEscherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.     

Nitration of tyrosine 92 mediates the activation of rat microsomal glutathione s-transferase by peroxynitrite

There is increasing evidence that protein function can be modified by nitration of tyrosine residue(s), a reaction catalyzed by proteins with peroxidase activity, or that occurs by interaction with peroxynitrite, a highly reactive oxidant formed by the reaction of nitric oxide with superoxide. Although there are numerous reports describing loss of function after treatment of proteins with peroxynitrite, we recently demonstrated that the microsomal glutathione S-transferase 1 is activated rather than inactivated by peroxynitrite and suggested that this could be attributed to nitration of tyrosine residues rather than to other effects of peroxynitrite. In this report, the nitrated tyrosine residues of peroxynitrite-treated microsomal glutathione S-transferase 1 were characterized by mass spectrometry and their functional significance determined. Of the seven tyrosine residues present in the protein, only those at positions 92 and 153 were nitrated after treatment with peroxynitrite. Three mutants (Y92F, Y153F, and Y92F, Y153F) were created using site-directed mutagenesis and expressed in LLC-PK1 cells. Treatment of the microsomal fractions of these cells with peroxynitrite resulted in an approximately 2-fold increase in enzyme activity in cells expressing the wild type microsomal glutathione S-transferase 1 or the Y153F mutant, whereas the enzyme activity of Y92F and double site mutant was unaffected. These results indicate that activation of microsomal glutathione S-transferase 1 by peroxynitrite is mediated by nitration of tyrosine residue 92 and represents one of the few examples in which a gain in function has been associated with nitration of a specific tyrosine residue.     

Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is a thiol-rich metallopeptidase ubiquitously distributed in mammalian tissues and involved in oligopeptide metabolism both within and outside cells. Fifteen Cys residues are present in the rat EP24.15 protein, seven are solvent accessible, and two are found inside the catalytic site cleft; no intraprotein disulfide is described. In the present investigation, we show that mammalian immunoprecipitated EP24.15 is S-glutathionylated. In vitro EP24.15 S-glutathionylation was demonstrated by the incubation of bacterial recombinant EP24.15 with oxidized glutathione concentration as low as 10 microM. The in vitro S-glutathionylation of EP24.15 was responsible for its oxidative oligomerization to dimer and trimer complexes. EP24.15 immunoprecipitated from cells submitted to oxidative challenge showed increased trimeric forms and decreased S-glutathionylation compared to immunoprecipitated protein from control cells. Our present data also show that EP24.15 maximal enzymatic activity is maintained by partial S-glutathionylation, a mechanism that apparently regulates the protein oligomerization. Present results raise the possibility of an unconventional property of protein S-glutathionylation, inducing oligomerization by interprotein thiol-disulfide exchange.     

A revised mechanism for the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA based on unexpected results with the C114A mutant

The compound (methylenecyclopropyl)formyl-CoA (MCPF-CoA) has been reported earlier as a potent active site-directed inactivator of bovine liver enoyl-CoA hydratase (ECH). It is believed that the mechanism of inactivation involves the attack of Cys114 at C-2' of MCPF-CoA, resulting in ring cleavage and permanent covalent modification of the enzyme. Here, we describe studies with the C114A mutant of bovine liver ECH, which was constructed and purified to determine the role of this residue in the catalytic mechanism of the enzyme. The C114A mutant, which is catalytically competent, shows an unexpected susceptibility to inactivation by MCPF-CoA, indicating that Cys114 is not the primary nucleophile responsible for the inactivation of the enzyme. To determine if catalytic residues Glu115 and Glu135 play a role in the inactivation of the enzyme, the E115Q and E135Q mutants were also constructed and purified. It was determined that these mutants did not react with MCPF-CoA, indicating a possible role for both residues in the inactivation of the wild-type enzyme. Pepsin digestion and subsequent LC-MS/MS analysis of the inactivated wild-type enzyme and C114A mutant revealed that Glu115 was modified in each case, supporting the hypothesis that this residue is the true nucleophile that traps MCPF-CoA and indicating that the covalent modification of Cys114 reported earlier may be a postinactivation artifact. We propose a modified mechanism of inactivation involving Glu115 and Glu135, and suggest that MCPF-CoA may be a mechanism-based inhibitor for bovine liver ECH.     

Oxidative processing of latent fas in the endoplasmic reticulum controls the strength of apoptosis

We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241-252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis.     

Role of cysteine residues in the activity of rat glutathione transferase P (7-7): elucidation by oligonucleotide site-directed mutagenesis

To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) form (GST-P), a cDNA clone, pGP5, containing the entire coding sequence of GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 13, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JM109. All four Cys residues in rGST-P were independently substituted with alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards 1-chloro-2,4-dinitrobenzene as liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intra- or inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity.     

Cellular Redox Imbalance and Changes of Protein S-glutathionylation Patterns Are Associated with Senescence Induced by Oncogenic H-Ras

H-Ras oncogene requires deregulation of additional oncogenes or inactivation of tumor suppressor proteins to increase cell proliferation rate and transform cells. In fact, the expression of the constitutively activated H-RasV12 induces cell growth arrest and premature senescence, which act like barriers in pre-neoplastic lesions. In our experimental model, human fibroblasts transfected with H-RasV12 show a dramatic modification of morphology. H-RasV12 expressing cells also show premature senescence followed by cell death, induced by autophagy and apoptosis. In this context, we provide evidence that in H-RasV12 expressing cells, the premature senescence is associated with cellular redox imbalance as well as with altered post-translation protein modification. In particular, redox imbalance is due to a strong reduction of total antioxidant capacity, and significant decrease of glutathione level. As the reversible addition of glutathione to cysteinyl residues of proteins is an important post-translational regulative modification, we investigated S-glutathionylation in cells expressing active H-Ras. In this contest we observed different S-glutathionylation patterns in control and H-RasV12 expressing cells. Particularly, the GAPDH enzyme showed S-glutathionylation increase and significant enzyme activity depletion in H-Ras V12 cells. In conclusion, we proposed that antioxidant defense reduction, glutathione depletion and subsequent modification of S-glutathionylation of target proteins contribute to arrest cell growth, leading to death of fibroblasts expressing constitutively active H-Ras oncogene, thus acting as oncogenic barriers that obstacle the progression of cell transformation.     

Crystallization and preliminary x-ray diffraction studies of a psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P2(1) and diffract to 2.1 A resolution. Form I has unit-cell parameters a=45.49A b=103.63A c=50.37A beta=108.2 degrees and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48A b=103.78A c=90.25A beta=103.8 degrees and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.     

Inactivation of NADP+-dependent isocitrate dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. We investigated whether the ICDH would be a vulnerable target of peroxynitrite anion (ONOO-) as a purified enzyme, in intact cells, and in liver mitochondria from ethanol-fed rats. Synthetic peroxynitrite and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a peroxynitrite-generating compound, inactivated ICDH in a dose- and time-dependent manner. The inactivation of ICDH by peroxynitrite or SIN-1 was reversed by dithiothreitol. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. Immunoblotting analysis of peroxynitrite-modified ICDH indicates that S-nitrosylation of cysteine and nitration of tyrosine residues are the predominant modifications. Using electrospray ionization mass spectrometry (ESI-MS) with tryptic digestion of protein, we found that peroxynitrite forms S-nitrosothiol adducts on Cys305 and Cys387 of ICDH. Nitration of Tyr280 was also identified, however, this modification did not significantly affect the activity of ICDH. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by peroxynitrite. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and binding of the hydrophobic probe 8-anilino-1-napthalene sulfonic acid. When U937 cells were incubated with 100 microM SIN-1 bolus, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitation and ESI-MS, we were also able to isolate and positively identify S-nitrosylated and nitrated mitochondrial ICDH from SIN-1-treated U937 cells as well as liver from ethanol-fed rats. Inactivation of ICDH resulted in the pro-oxidant state of cells reflected by an increased level of intracellular reactive oxygen species, a decrease in the ratio of [NADPH]/[NADPH + NADP+], and a decrease in the efficiency of reduced glutathione turnover. The peroxynitrite-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

S-glutathionylation of human glyceraldehyde-3-phosphate dehydrogenase and possible role of Cys152-Cys156 disulfide bridge in the active site of the protein

BackgroundWe previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H₂O₂ and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated.MethodsHuman recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH.ResultsS-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S.ConclusionsS-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1.General significanceThe described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.     

Cysteine is the presumed catalytic residue of Citrus sinensis phospholipid hydroperoxide glutathione peroxidase over‐expressed under salt stress

A citrus salt‐stress associated protein (Cit‐SAP), partially purified from citrus cultured cells, was previously identified as the first plant phospholipid hydroperoxide glutathione peroxidase (PHGPx). The nucleotide sequence of its isolated gene ( csa ) revealed that a TGT, known as codon for Cys, encodes the presumed catalytic residue 41 in the polypeptide chain of Cit‐SAP. In animals, a TGA encodes the rare amino acid selenocysteine (Sec) as the catalytic residue of the analogous enzyme. It is of interest to establish whether the TGT codon for this catalytic residue in the plant enzyme is indeed translated to Cys and not to Sec, and to demonstrate the effect of such a change, if it exists, on the nature of the enzymatic activity of the plant enzyme as compared to that of the animal. In the present study, we have purified for the first time, by affinity chromatography, enzymatically active citrus PHGPx from recombinant Escherichia coli bearing the csa gene. Tryptic digestion of the purified enzyme followed by HPLC afforded the isolation of a peptide which contains residue 41, and its sequence analysis revealed that this residue is indeed a Cys, and not Sec. The enzymatic activity and specificity of the recombinant Cit‐SAP was found to be similar to that observed before for the partially purified plant enzyme. However, the rate of this activity was much lower towards phospholipid hydroperoxides, and none towards hydrogen peroxide, as compared to that of the animal analogue. It is therefore suggested that the presence of a Cys, and not Sec, as the catalytic residue in the plant enzyme, affects its enzymatic activity and may determine a different biological role for the plant PHGPx from that of the animal.     

Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase.

Skeletal muscle contraction and relaxation is efficiently modulated through the reaction of reactive oxygen-nitrogen species with sarcoplasmic reticulum protein thiols in vivo. However, the exact locations of functionally important modifications are at present unknown. Here, we determine by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity. Despite the size and nature of the SR Ca-ATPase, a 110 kDa membrane protein, identification and quantitation of Cys modification was achieved through labeling with 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABMI) and/or N-(2-iodoethyl)trifluoroacetamide (IE-TFA) followed by an exhaustive tryptic digestion and on-line HPLC-UV-electrospray MS analysis. The reaction with IE-TFA generates aminoethylcysteine, a new trypsin cleavage site, which allows the production of specific peptide fragments that are diagnostic for IE-TFA labeling, conveniently identified by mass spectrometry. Exposure of the SR Ca-ATPase to low concentrations (0.1 mM) of peroxynitrite resulted in the fully reversible chemical modification of Cys at positions 344, 349, 471, 498, 525, and 614 (nitrosylation of Cys(344) and Cys(349) was seen), whereas higher concentrations of peroxynitrite (0.45 mM) additionally affected Cys residues at positions 636, 670, and 674. When the SR Ca-ATPase was exposed to 0.45 mM peroxynitrite in the presence of 5.0 mM glutathione (GSH), thiol modification became partially reversible and S-glutathiolation was detected for Cys residues at positions 344, 349, 364, 498, 525, and 614. The extent of enzyme inactivation (determined previously) quantitatively correlated with the loss of labeling efficiency (i) of a single Cys residue and (ii) of the tryptic fragment containing both Cys(344) and Cys(349). Earlier results had shown that the independent selective modification of Cys(344) is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo) 102, 103-109]. Thus, we conclude that modification of only Cys(349) is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.     

Peroxynitrite irreversibly inactivates the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) in human breast cancer cells: a cellular and mechanistic study

Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (k(inact) = 5 x 10(4) m(-1).s(-1)) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1.     

Human glutathione transferase P1-1 and nitric oxide carriers; a new role for an old enzyme

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i) < 10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.     

Chloroplast fructose 1,6-bisphosphatase with changed redox modulation: comparison of the Galdieria enzyme with cysteine mutants from spinach

Spinach fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), a redox-modulated chloroplast enzyme and part of the Calvin cycle, and three different Cys mutants were expressed in E. coli. The properties of the purified proteins were compared to those of native and recombinant chloroplast FBPase from the red alga Galdieria sulphuraria. In spinach chloroplast FBPase, Cys(155) and Cys(174) are engaged in the formation of the disulfide bridge. The corresponding mutants are active when expressed in E. coli, while C179S is inactive and can be reductively activated as can the wild-type enzyme. The active C174S mutant, however, could be inactivated by oxidation, and reactivated, but only by reduction, not alternatively with high pH and high Mg(2+) as is the case for the wild-type enzyme. In the sequence of Galdieria FBPase, the Cys that corresponds to Cys(179) in the spinach enzyme is lacking. However, the Galdieria FBPase, in contrast to the spinach Cys(179) mutant, does not show any indication for a comparable redox modulation of its activity. Instead, oxidation only leads to partial inactivation without any qualitative changes in enzyme properties. Upon reduction, the lost activity can be recovered.