The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn2+ (up to 2 mM) and activated above this concentration. Ca2+ inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn^{2 +} (up to 2 mM) and activated above this concentration. Ca^{2 +} inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Comparative in vitro effects of some metal ions on bovine kidney cortex glutathione reductase

Heavy metal pollution can arise from many sources and damage many organisms. Exposure to the metal ions can leads to a reduction in cellular antioxidant enzyme activities and lowers cellular defense against oxidative stress. In this study we have tested effects of the some metal ions on the purified bovine kidney cortex glutathione reductase (GR). Cadmium (Cd2+), nickel (Ni2+), and zinc (Zn2+) showed inhibitory effect on the enzyme. The obtained IC?? values of Cd2+, Ni2+, and Zn2+ are 0.027, 0.8, and 1 mM, respectively. Kinetic characterization of the inhibition is also investigated. Cd2+ inhibition is noncompetitive with respect to both oxidized glutathione (GSSG) (Ki(GSSG) 0.060 ± 0.005 mM) and NADPH (Ki(NADPH) 0.025 ± 0.002 mM). Ni2+ inhibition is noncompetitive with respect to GSSG (Ki(GSSG) 0.329 ± 0.016 mM) and uncompetitive with respect to NADPH (Ki(NADPH) 0.712 ± 0.047 mM). The effect of Zn2+ on GR activity is consistent with noncompetitive inhibition pattern when the varied substrate is the GSSG (Ki(GSSG) 0.091 ± 0.005 mM) and the NADPH (Ki(NADPH) 0.226 ± 0.01 mM), respectively. GR inhibition studies may be useful for understanding the mechanisms for oxidative damage associated with heavy metal toxicity.     

Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

Glutathione reductase from rat liver has been purified greater than 5000-fold in a yield of 20%. The molecular weights of the enzyme and its subunits were estimated to be 125,000 and 60,000, respectively, indicating that the native enzyme is a dimer. The enzyme molecular contains 2 FAD molecules, which are reducible by NADPH, GSH or dithioerythritol. The reduced flavin is instantaneously reoxidized by addition of GSSG. The steady state kinetic data are consistent with a branching reaction mechanism previously proposed for glutathione reductase from yeast (MANNERVIK, B. (1973) Biochem. Biophy. Res. Commun. 53, 1151-1158). This mechanism is also favored by the nonlinear inhibition pattern produced by NADP-+. However, at low GSSG concentrations the rate equation can be approximated by that of a simple ping pong mechanism. NADPH and the mixed disulfide of coenzyme A and GSH were about 10% as active as NADPH and GSSG, respectively, whereas some sulfenyl derivatives related to GSSG were less active as substrates. The pH activity profiles of these substrates differed from that of the NADPH-GSSG substrate pair.     

Mechanistic studies on a novel, highly potent gold-phosphole inhibitor of human glutathione reductase

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When approximately 1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a K(d)((app)) for GSSG of 63 microm, and a k((obs)max) of 106 s(-1) at 4 degrees C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (K(d) = 0.46 microm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range.     

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD⁺ and NADPH/NADP⁺. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].     

Characterization of the inhibition effect induced by nickel on glucose-6-phosphate dehydrogenase and glutathione reductase

Kinetic characterization of the inhibition effect of nickel on glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G-6-PD) and glutathione reductase (GR; EC 1.6.4.2) from Saccharomyces cerevisiae was made. The effect of nickel on G-6-PD activity is consistent with a mixed-type inhibition pattern, with a competitive character, since the inequality ki,int greater than ki,slope shows an inverse relation between varied substrate concentrations and fractional inhibition. An inhibition effect of nickel on GR activity, when NADPH is the varied substrate, is also consistent with a mixed-type inhibition pattern. However, pure competitive inhibition is found on GR reaction when oxidized glutathione is the varied substrate. This investigation shows the highest sensibility of GR before the inhibitory effect of nickel, in agreement with the experimental values of inhibition constants found in this study, where constants related to the GR system are lower than the ones of the G-6-PD system.     

A study of the kinetic mechanism followed by glutathione reductase from mycelium of Phycomyces blakesleeanus

An investigation of the reaction mechanism of glutathione reductase isolated from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) was conducted. The enzyme showed GSSG concentration-dependent substrate inhibition by NADPH and pH-dependent substrate inhibition by GSSG. At pH 7.5, the kinetic data were consistent with a basic scheme corresponding to the branching mechanism, involving a ping-pong with formation of a dead-end F.NADPH complex and an ordered sequential mechanism. Both pathways have in common the step in which NADPH binds to the free oxidized form (E) of the glutathione reductase. At low concentrations of GSSG the ping-pong mechanism prevails, whereas at high concentrations the ordered mechanism appears to dominate. The data were analyzed on the basis of the limiting ping-pong mechanism with F.NADPH complex formation and of the hybrid mechanism, and the kinetic constants of the model were calculated. The data obtained at acidic pH values do not rule out the possibility that the kinetic model may be more complicated than the basic scheme studied.     

Role of glutathione reductase system in disulfiram conversion to diethyldithiocarbamate.

Experiments were carried out to establish the role of glutathione reductase (GR), if any, in the metabolic conversion of disulfiram (DS) to diethyldithiocarbamate (DDC). It was observed that, under standard assay conditions, whereas DS was incorporated as a substrate instead of oxidised glutathione (GSSG), the enzymes from both human liver extract and yeast sources failed to reduce the parent compound, implying that glutathione reductase perse do not reduce disulfiram. However, the incorporation of disulfiram into an assay system comprising of GSSG, NADPH and reductase resulted in DS reduction to DDC. Further, the observation, that the GR assay system devoid of either GSSG or NADPH was found to lack DS reducing ability, implies that GSH as a reaction product of GR system is responsible for the reduction of DS to DDC. The results of in-vitro experiments indicated that GSH perse could reduce DS to DDC nonenzymatically, with a stoichiometric relationship of 2:1. Thus it is inferred that GR perse do not reduce DS, whereas GSH, as an intermediary metabolite of GR system, brings about non-enzymatic reduction of DS via a sulfhydral group exchange reaction.     

Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity

The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.     

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.     

Reductive inactivation of yeast glutathione reductase by Fe(II) and NADPH

Glutathione reductase (GR) carries out the enzymatic reduction of glutathione disulfide (GSSG) to its reduced form (GSH) at the expense of the reducing power of NADPH. Previous studies have shown that GR from several species is progressively inactivated in the presence of NADPH, but that the mechanism of inactivation (especially in the presence of metals) has not been fully elucidated. We have investigated the involvement of iron ions in the inactivation of yeast (Saccharomyces cerevisiae) GR in the presence of NADPH. Even in the absence of added iron, inactivation of GR was partly blocked by the iron chelators, deferoxamine and ortho-phenanthroline, suggesting the involvement of trace amounts of contaminating iron in the mechanism of inhibition. Exogenously added antioxidants including ethanol, dimethylsulfoxide and 2-deoxyribose did not protect GR against NADPH-induced inactivation, whilst addition of exogenous Fe(II) (but not Fe(III)) potentiated the inactivation. Moreover, removal of oxygen from the medium led to increased inhibition of GR, whereas pre-incubation of the Fe(II)-containing medium for 30 min under normoxic conditions prior to the addition of GR abolished the enzyme inactivation by NADPH. Under these pre-incubation conditions, Fe(II) is fully oxidized to Fe(III) within 1 min. Furthermore, GR that had been previously inactivated in the presence of Fe(II) plus NADPH could be partially reactivated by treatment with ortho-phenanthroline and deferoxamine. In contrast, Fe(III) had no effect on GR reactivation. Together, these results indicate that yeast GR is inactivated by a reductive mechanism mediated by NADPH and Fe(II). According to this mechanism, GR is diverted from its normal redox cycling by the generation of an inactive reduced enzyme form in which both the FAD and thiol groups at the active site are likely in a reduced state.     

Glutathione reductase in the sea urchin egg. III. Activation of the complex form by proteinases.

The G-200 flow-through fraction of the extract of sea urchin eggs contained a complex form of glutathione reductase (GR) [EC 1.6.4.2]. The complex was unstable and gradually dissociated with ain increase in GR activity. The activation was facilitated by high concentrations of EDTA, KCI or (NH4)2SO4. The rate of activation by salts was apparently dependent on the ionic strength. The complex form was also activated rather quickly by treatment with proteinases such as trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1] or subtilisin [EC 3.4.21.14]. Trypsin caused the complex to release the free form of GR. Even after trypsin treatment, little change was observed in the dependence of the GR activity on GSSG or NADPH concentration. The GR activity of the complex form was not inhibited at all by 0.2 mM N-ethylmaleimide (NEM) in the presence of GSSG, but was reduced to 3% in the presence of NADPH. When excess NEM was sequestered with GSH, the NEM-treated complex form was strikingly activated by trypsin, while no activation was detected with the free form of enzyme pretreated with NEM. These results suggest that the active site of GR in the complex form is largely masked by a polypeptide moiety of theinhbitiory component.     

Wheat Chloroplastic Glutathione Reductase Activity is Regulated by the Combined Effect of pH, NADPH and GSSG

To study the mechanism of regulation of chloroplastic glutathionereductase (GR) under photooxidative conditions, GR activity,and the levels of NADPH, GSH and GSSG were measured in wheatchloroplasts under photooxidative light. GR was extremely labile,and the concentrations of GSH and GSSG progressively diminishedin chloroplasts prepared without ascorbate. The NADPH leveldid not significantly change during photooxidative treatment.The addition of 10 mM ascorbate to the incubation medium preventedthe decrease of GSH and GSSG and strongly protected GR activity.However, ascorbate had no effect on NADPH-dependent inhibitionof the chloroplastic GR purified from wheat leaves. We studiedthe effect of NADPH, temperature, pH and GSSG on the purifiedenzyme. The inhibition by NADPH was greatly dependent on temperatureand pH. NADPH inhibited GR by around 93% up to pH 7.5, but withina range of 8.0 to 9.5 the inhibition was only marginal. ThepH dependence of the NADPH inhibitory effect could be due, atleast in part, to different rates in the generation of NADPH-X,a derivative of NADPH which inactivates several pyridin nucleotidedehydrogenases. Furthermore, the NADPH-dependent inhibitionwas almost completely prevented by GSSG, but not by GSH. (Received July 9, 1998; Accepted April 30, 1999)     

Evidence for a role of a vicinal dithiol in catalysis and proton pumping in mitochondrial nicotinamide nucleotide transhydrogenase

The effect of glutathione, glutathione disulfide and the dithiol reagent phenylarsine oxide on purified soluble as well as reconstituted mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. Glutathione disulfide and phenylarsine oxide caused an inhibition of transhydrogenase, the extent of which was dependent on the presence of either of the transhydrogenase substrates. In the absence of NADPH glutathione protected partially against inactivation by glutathione disulfide and phenylarsine oxide. In the presence of NADPH glutathione also inhibited transhydrogenase. Reconstituted transhydrogenase vesicles behaved differently as compared to the soluble transhydrogenase and was partially uncoupled by GSSG. It is concluded that transhydrogenase contains a dithiol that is essential for catalysis as well as for proton translocation.     

Kinetic characterization of glutathione reductase from the malarial parasite Plasmodium falciparum. Comparison with the human enzyme

The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG + NADPH + H(+) <--> 2 GSH + NADP(+)). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative half-reaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E(ox)-EH(2) and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K(eq) from the Haldane relationship, 0.075, very similar to the K(eq) of the same reaction for the yeast enzyme (0.085) (Arscott, L. D., Veine, D. M., and Williams, C. H., Jr. (2000) Biochemistry 39, 4711-4721). Enzyme-monitored turnover indicates that E(FADH(-))(S-S). NADP(+) and E(FAD)(SH)(2).NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance.     

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.     

Inhibition of glutathione reductase by isoproterenol oxidation products

Oxidative stress induced by catecholamines is a well recognized toxic event. This effect has been extensively observed in the heart, where high levels of catecholamines cause enzyme inhibition, lipid peroxidation, energy depletion and myocardial necrosis. Catecholamines can be converted into o-quinones and undergo cyclization into aminochromes. This process can occur enzymatically or through autoxidation and involves the formation of free radicals. Aminochromes are highly reactive molecules that can cause oxidation of protein sulfhydryl groups and deamination catalysis, among other deleterious effects; in addition, inhibition of some enzymes has been also reported. We have studied the effects of isoproterenol oxidation products (IOP) on glutathione reductase (GR) activity in vitro. Isoproterenol (ISO) autoxidation was conducted at 37 degrees C in the dark, for 4 h at pH 7.0 and this process was monitored by UV spectrophotometry at both 340 and 490nm. Addition of the autoxidized solution to GR in the presence of oxidized glutathione (GSSG) and NADPH showed that IOP inhibits GR in a competitive mode and that this effect increases during the 4 h incubation period. This inhibitory effect of IOP was partially prevented by the addition of reduced glutathione (GSH), L-cysteine and ascorbic acid to the reaction mixtures.