Structure analysis and molecular model of the selenoenzyme glutathione peroxidase at 2.8 Å resolution

The three-dimensional structure of bovine erythrocyte glutathione peroxidase, a tetrameric enzyme containing 4 gram atoms of selenium per mole (M_r = 84,000), has been determined at 2.8 Å resolution using the multiple isomorphous replacement method. By correlation calculations in Patterson space the tetramers were shown to exhibit molecular [222] symmetry, proving the monomers to be identical or at least very similar.The monomer consists of a single polypeptide chain of 178 amino acid residues. Its shape is nearly spherical with a radius of $\text{r ≈ 19}\text{A}\text{?}$. A tentative sequence corresponding to a partially refined model (R = 0.38) is given. Each subunit is built up from a central core of two parallel and two anti-parallel strands of pleated sheet surrounded by four α-helices. One of the helices runs antiparallel to the neighbouring β-strands giving rise to a βαβ substructure, an architecture that has been found in several other proteins e.g. flavodoxin, thioredoxin, rhodanese and dehydrogenases. A comparison of the glutathione peroxidase subunit structure with thioredoxin-S₂ revealed large regions of structural resemblance. The central four-stranded β structure together with two parallel α-helices resembles nearly 80% of the thioredoxin fold.The active sites of glutathione peroxidase are located in flat depressions on the molecular surface. Probably each active centre is built up by segments from two subunits. The catalytically active selenocysteines were found at the N-terminal ends of long α-helices and are surrounded by an accumulation of aromatic side-chains. A difference Fourier map between oxidized and substrate-reduced glutathione peroxidase as well as heavy-atom binding led to the conclusion that the two-electron redox-cycle involves a reversible transition of the active-site selenium from a selenenic acid (RSeOH) to a seleninic acid (RSeOOH).     

Zinc toxicity in various lung cell lines is mediated by glutathione and GSSG reductase activity

In a previous work, it was shown that in cells after a decrease of cellular glutathione content, toxic zinc effects, such as protein synthesis inhibition or GSSG (glutathione, oxidized form) increases, were enhanced. In this study, zinc toxicity was determined by detection of methionine incorporation as a parameter of protein synthesis and GSSG increase in various lung cell lines (A549, L2, 11Lu, 16Lu), dependent on enhanced GSSG reductase activities and changed glutathione contents. After pretreatment of cells with dl-buthionine-[R,S]-sulfoximine (BSO) for 72 h, cellular glutathione contents were decreased to 15–40% and GSSG reductase activity was increased to 120–135% in a concentration-dependent manner. In BSO pretreated cells, the IC₅₀ values of zinc for methionine incorporation inhibition were unchanged as compared to cells not pretreated. The GSSG increase in BSO pretreated cells by zinc was enhanced in L2, 11Lu, and 16Lu cells, whereas in A549 cells, the GSSG increase by zinc was enhanced only after pretreatment with the highest BSO concentration. Inhibition of GSSG reductase in alveolar epithelial cells was observed at lower zinc concentrations than needed for methionine incorporation inhibition, whereas in fibroblastlike cells, inhibition of GSSG reductase occurred at markedly higher zinc concentrations as compared to methionine incorporation inhibition. These results demonstrate that GSSG reductase is an important factor in cellular zinc susceptibility. We conclude that reduction of GSSG is reduced in zinc-exposed cells. Therefore, protection of GSH oxidation by various antioxidants as well as enhancement of GSH content are expected to be mechanisms of diminishing toxic cellular effects after exposure to zinc.     

Electron transfer properties of melanin.

Melanin synthesized from mushroom tyrosinase and 3,4-dihydroxyphenylalanine has been shown to oxidize NADH and NADPH, reduce ferricyanide, oxidized forms of cytochrome c and dichlorophenolindophenol, and catalyze the coupled oxidation of NADH and reduction of ferricyanide. Kinetic studies involving the determination of initial velocity at various concentrations of substrates and product inhibition measurements have been carried out on the NADH-ferricyanide-melanin reaction. The results are consistent with a ping-pong mechanism in which one product is formed prior to the reaction of melanin with the second substrate involving the reversible oxidation and reduction of melanin during the reaction. It may be concluded that melanin is capable of acting as an electron transfer agent in several reduction-oxidation systems.     

Studies on glutathione transport utilizing inside-out vesicle prepared from human erythrocytes

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Line-weaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25°C, this transport activity was easily lost during the storage of vesicles at 4°C. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation of transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37°C, this transport activity was stable during storage of vesicles at 4°C for several days. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.     

Status of reduced glutathione in primary cultures of rat hepatocytes and the effect on conjugation of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide

The intracellular level of reduced glutathione (GSH) and GSH conjugation have been investigated in primary cell cultures of hepatocytes isolated from control rats, phenobarbitone (PB) and 3-methylcholanthrene (MC) treated rats. The data demonstrate that in all cell cultures the GSH concentrations show a triphasic pattern: (i) within 1 h of culture an initial marked decrease to 50% of the levels found in fresh hepatocytes; (ii) recovery of GSH concentrations to above the levels observed in fresh cells. This occurs after 6 h in culture with control cells and after 10-24 h with cells from either PB or MC treated rats and was most prominent in cells from PB-treated rats. (iii) A slow decline to between 30 and 40 nmol GSH/mg protein from 24 to 96 h in culture. Synthesis of GSH was slower in cultured cells from PB treated rats and this was confirmed by the resynthesis rates when diethylmaleate (DEM) was used to deplete GSH. The formation of GSH conjugates with racemic 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was measured in control cells in suspension and after 3 and 24 h in culture. Despite the decrease in GSH concentrations observed between 1 and 4 h after culture, the conjugation rates were not decreased.     

Covalent attachment of 4-thiouridylic acid to ribonuclease A by near-ultraviolet radiation.

Bovine pancreatic ribonuclease A (EC.2.7.7.16) was irradiated with near-ultraviolet light (334 and 365 nm) in the presence of equimolar amount of a substrate analog 4-thio[¹⁴C]uridine 3′-phosphate. Gel-filtration studies revieled that one to two moles of the nucleotide entered into covalent attachment to the enzyme under either aerobic or anaerobic irradiation. Reduction with dithiothreitol of the irradiated protein released about one-third of the attached materials. A model experiment with oxidized glutathione and radioactive 4-thiouridine suggested the formation of aducts between cystinyl residue and the pyrimidine base. The covalent attachment of nucleotide to ribonuclease was independent of inactivation of the enzyme.     

DehydroalanylGly,a new post translational modification resulting from the breakdown of glutathione

Background

The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues.

Some observations on the chemical and stereochemical specificity of the de-alkylation of organophosphorus esters by a hepatic glutathione transferase

Hepatic glutathione (GSH) S-methyl transferase from rabbit, pig and dog demethylates dimethyl phosphate triesters. No stereospecificity towards racemic ethyl methyl 2-chloro-1-(2,4-dichlorophenyl)vinyl phosphate could be demonstrated but the enzyme exhibited some selectivity towards the (+) and (?) forms of O-methyl S-methyl 1-naphthyl phosphorothiolate. Pig liver enzyme, purified 30-fold, demethylated the prochiral substrate dimethyl 1-naphthyl phosphorothionate with 90% stereo-selectivity.     

Highly potent and specific inhibitors of human renin

We designed aldehyde derivatives of small peptides representing the C-terminal portion of angiotensin I sequence as an inhibitor of human renin. Among compounds that we synthesized, benzyloxycarbonyl (Z)-Phe-His-Leucinal (compound V), Z-Pro-Phe-His-Leucinal (Compound IV) and Z-[3-(1'-naphthyl)Ala]-His-Leucinal (compound VII) markedly inhibited human renin (IC50, 7.5 X 10(-7), 3.2 X 10(-7) and 8.0 X 10(-8) mol/l, respectively). Compound VII was shown to be noncompetitive (Ki = 2.4 X 10(-7) mol/l). It did not inhibit either cathepsin D or pepsin. Compound V had slight or no inhibitory effect at the concentration of 10(-5) mol/l on six animal renins except for monkey and rabbit renins. Results obtained show that these aldehyde compounds are highly selective and species specific inhibitors for human and monkey renins.     

Glutathione redox system,GSH-Px activity and lipid peroxidation (LPO) levels in tadpoles of R.r.ridibunda and B.viridis

Total glutathione (t-GSH), reduced glutathione (GSH), glutathione disulphide (GSSG) levels, t-GSH/GSSG ratio, glutathione peroxidase (GSH-Px) activity and lipid peroxidation (LPO) levels were investigated during the development period of a predominantly aquatic amphibian R.r.ridibunda and a predominantly terrestrial amphibian B. viridis. While t-GSH and GSH showed a similar trend, GSSG concentration increased significantly (p<0.05) during the larval stages in R.r.ridibunda larvae. In contrast to R.r.ridibunda larvae, there was no significant (p>0.05) change between 1 and 5 weeks in the t-GSH and GSH concentrations of B. viridis. t-GSH and GSH concentrations of B. viridis larvae became sharply elevated after the fifth week, GSSG levels increased 3.25-fold during the metamorphosis. The t-GSH/GSSG ratio fluctuated and the lowest t-GSH/GSSG ratios were observed at the third week for both species. GSH-Px activities for both species increased significantly (p<0.05) during the growing period. The highest GSH-Px activities in R.r.ridibunda and B.viridis were observed at the eighth week and they were 3.45 +/- 0.17 and 4.1 +/- 0.21 IU mg(-1), respectively. The membrane LPO levels in the R.r.ridibunda and B. viridis tadpoles significantly (p<0.001) decreased from 206 +/- 10.3 to 146 +/- 7.3 and from 198 +/- 9.9 to 23 +/- 1.15 nmol MDA g(-1) w.w., respectively.     

The function of gamma-glutamyl transpeptidase as a determinant in cell sensitivity to azaserine toxicity

The enzyme gamma-glutamyl transpeptidase (GGT) is characteristically present at high levels in mammalian cells that are vulnerable in vivo to the selectively toxic and carcinogenic effects of the naturally occurring diazo amino acid L-azaserine. The possible role of GGT as a determinant of cellular sensitivity to azaserine toxicity was investigated. No correlation was found between GGT activity and the abilities of different cell lines or GGT-deficient cell strains of TuWi, a human nephroblastoma-derived line high in GGT, to accumulate azaserine. However, the thiols glutathione and cysteine were found to inhibit the toxicity of azaserine in cultures of TuWi. In addition, maleate lowered both intracellular and extracellular glutathione levels and enhanced sensitivity of TuWi cells to azaserine, while serine-borate, a potent inhibitor of GGT, increased extracellular glutathione levels and inhibited azaserine toxicity. Since extracellular glutathione accumulation, which may reflect the rate of cellular glutathione turnover, is increased in cultures of azaserine-resistant, GGT-deficient strains of TuWi, we propose that GGT enhances cellular sensitivity to azaserine primarily by increasing the rate of glutathione turnover, thus removing the glutathione from detoxification pathways.     

Molecular cloning and characterization of the glutathione reductase gene from Stipa purpurea

Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.     

Transient light-induced intracellular oxidation revealed by redox biosensor

We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.     

The Zinc Center Influences the Redox and Thermodynamic Properties of Escherichia coli Thioredoxin 2

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-terminal zinc-binding domain. We have determined the redox potential of Trx2, the pK_a of the active site nucleophilic cysteine, as well as the stability of the oxidized and reduced form of the protein. Trx2 is more oxidizing than Trx1 (-221 mV versus -284 mV, respectively), which is in good agreement with the decreased value of the pK_a of the nucleophilic cysteine (5.1 versus 7.1, respectively). The difference in stability between the oxidized and reduced forms of an oxidoreductase is the driving force to reduce substrate proteins. This difference is smaller for Trx2 (ΔΔG°_H2O = 9 kJ/mol and ΔT_m = 7. 4 °C) than for Trx1 (ΔΔG°_H2O = 15 kJ/mol and ΔT_m = 13 °C). Altogether, our data indicate that Trx2 is a significantly less reducing enzyme than Trx1, which suggests that Trx2 has a distinctive function. We disrupted the zinc center by mutating the four Zn²⁺-binding cysteines to serine. This mutant has a more reducing redox potential (-254 mV) and the pK_a of its nucleophilic cysteine shifts from 5.1 to 7.1. The removal of Zn²⁺ also decreases the overall stability of the reduced and oxidized forms by 3.2 kJ/mol and 5.8 kJ/mol, respectively. In conclusion, our data show that the Zn²⁺-center of Trx2 fine-tunes the properties of this unique thioredoxin.     

Understanding nicotinamide dinucleotide cofactor and substrate specificity in class I flavoprotein disulfide oxidoreductases: crystallographic analysis of a glutathione amide reductase

Glutathione reductase (GR) plays a vital role in maintaining the antioxidant levels of the cytoplasm by catalyzing the reduction of glutathione disulfide to reduced glutathione, thereby using NADPH and flavin adenine dinucleotide as cofactors. Chromatiaceae have evolved an unusual homolog that prefers both a modified substrate (glutathione amide disulfide [GASSAG]) and a different cofactor (NADH). Herein, we present the crystal structure of the Chromatium gracile glutathione amide reductase (GAR) both alone and in complex with NAD⁺. An altered charge distribution in the GASSAG binding pocket explains the difference in substrate specificity. The NADH binding pocket of GAR differs from that of wild-type GR as well as that of a low active GR that was engineered to mimic NADH binding. Based on the GAR structure, we propose two attractive rationales for producing an efficient GR enzyme with NADH specificity.