Balázs Németi

Mechanism of Thiol-Supported Arsenate Reduction Mediated by Phosphorolytic-Arsenolytic Enzymes II. Enzymatic Formation of Arsenylated Products Susceptible for Reduction to Arsenite by Thiols

Enzymes catalyzing the phosphorolytic cleavage of their substrates can reduce arsenate (AsV) to the more toxic arsenite (AsIII) via the arsenolytic substrate cleavage in presence of a reductant, as glutathione or dithiotreitol (DTT). We have shown this for purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen phosphorylase-a (GPa), and phosphotransacetylase (PTA). Using a multidisciplinary approach, we explored the mechanism whereby these enzymes mediate AsV reduction. It is known that PNP cleaves inosine with AsV into hypoxanthine and ribose-1-arsenate. In presence of inosine, AsV and DTT, PNP mediates AsIII formation. In this study, we incubated PNP first with inosine and AsV, allowing the arsenolytic reaction to run, then blocked this reaction with the PNP inhibitor BCX-1777, added DTT and continued the incubation. Despite inhibition of PNP, large amount of AsIII was formed in these incubations, indicating that PNP does not reduce AsV directly but forms a product (i.e., ribose-1-arsenate) that is reduced to AsIII by DTT. Similar studies with the other arsenolytic enzymes (GPa, GAPDH, and PTA) yielded similar results. Various thiols that differentially supported AsV reduction when present during PNP-catalyzed arsenolysis (DTT approximately dimercaptopropane-1-sulfonic acid > mercaptoethanol > DMSA > GSH) similarly supported AsV reduction when added only after a transient PNP-catalyzed arsenolysis, which preformed ribose-1-arsenate. Experiments with progressively delayed addition of DTT after BCX-1777 indicated that ribose-1-arsenate is short-lived with a half-life of 4 min. In conclusion, phosphorolytic enzymes, such as PNP, GAPDH, GPa, and PTA, promote thiol-dependent AsV reduction because they convert AsV into arsenylated products reducible by thiols more readily than AsV. In support of this view, reactivity studies using conceptual density functional theory reactivity descriptors (local softness, nucleofugality) indicate that reduction by thiols of the arsenylated metabolites is favored over AsV.

Polynucleotide Phosphorylase and Mitochondrial ATP Synthase Mediate Reduction of Arsenate to the More Toxic Arsenite by Forming Arsenylated Analogues of ADP and ATP

We have demonstrated that phosphorolytic-arsenolytic enzymes can promote reduction of arsenate (AsV) into the more toxic arsenite (AsIII) because they convert AsV into an arsenylated product in which the arsenic is more reducible by glutathione (GSH) or other thiols to AsIII than in inorganic AsV. We have also shown that mitochondria can rapidly reduce AsV in a process requiring intact oxidative phosphorylation and intramitochondrial GSH. Thus, these organelles might reduce AsV because mitochondrial ATP synthase, using AsV instead of phosphate, arsenylates ADP to ADP-AsV, which in turn is readily reduced by GSH. To test this hypothesis, we first examined whether the RNA-cleaving enzyme polynucleotide phosphorylase (PNPase), which can split poly-adenylate (poly-A) by arsenolysis into units of AMP-AsV (a homologue of ADP-AsV), could also promote reduction of AsV to AsIII in presence of thiols. Indeed, bacterial PNPase markedly facilitated formation of AsIII when incubated with poly-A, AsV, and GSH. PNPase-mediated AsV reduction depended on arsenolysis of poly-A and presence of a thiol. PNPase can also form AMP-AsV from ADP and AsV (termed arsenolysis of ADP). In presence of GSH, this reaction also facilitated AsV reduction in proportion to AMP-AsV production. Although various thiols did not influence the arsenolytic yield of AMP-AsV, they differentially promoted the PNPase-mediated reduction of AsV, with GSH being the most effective. Circumstantial evidence indicated that AMP-AsV formed by PNPase is more reducible to AsIII by GSH than inorganic AsV. Then, we demonstrated that AsV reduction by isolated mitochondria was markedly inhibited by an ADP analogue that enters mitochondria but is not phosphorylated or arsenylated. Furthermore, inhibitors of the export of ATP or ADP-AsV from the mitochondria diminished the increment in AsV reduction caused by adding GSH externally to these organelles whose intramitochondrial GSH had been depleted. Thus, whereas PNPase promotes reduction of AsV by incorporating it into AMP-AsV, the mitochondrial ATP synthase facilitates AsV reduction by forming ADP-AsV; then GSH can easily reduce these arsenylated nucleotides to AsIII.

Mechanism of thiol-supported arsenate reduction mediated by phosphorolytic-arsenolytic enzymes: I. The role of arsenolysis

Several mammalian enzymes catalyzing the phosphorolytic-arsenolytic cleavage of their substrates (thus yielding arsenylated metabolites) have been shown to facilitate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in presence of their substrate and a thiol. These include purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycogen phosphorylase-a (GPa). In this work, we tested further enzymes, the bacterial phosphotransacetylases (PTAs) and PNP, for AsV reduction. The PTAs, which arsenolytically cleave acetyl-CoA producing acetyl-arsenate, were compared with GAPDH, which can also form acetyl-arsenate by arsenolysis of its nonphysiological substrate, acetyl-phosphate. As these enzymes also mediated AsV reduction, we can assert that facilitation of thiol-dependent AsV reduction may be a general property of enzymes that catalyze phosphorolytic-arsenolytic reactions. Because with all such enzymes arsenolysis is obligatory for AsV reduction, we analyzed the relationship between these two processes in presence of various thiol compounds, using PNP. Although no thiol influenced the rate of PNP-catalyzed arsenolysis, all enhanced the PNP-mediated AsV reduction, albeit differentially. Furthermore, the relative capacity of thiols to support AsV reduction mediated by PNP, GPa, PTA, and GAPDH apparently depended on the type of arsenylated metabolites (i.e., arsenate ester or anhydride) produced by these enzymes. Importantly, AsV reduction by both acetyl-arsenate-producing enzymes (i.e., PTA and GAPDH) exhibited striking similarities in responsiveness to various thiols, thus highlighting the role of arsenylated metabolite formation. This observation, together with the finding that PNP-mediated AsV reduction lags behind the PNP-catalyzed arsenolysis lead to the hypothesis that arsenolytic enzymes promote reduction of AsV by forming arsenylated metabolites which are more reducible to AsIII by thiols than inorganic AsV. This hypothesis is evaluated in the adjoining paper.

Reduction of Dimethylarsinic Acid to the Highly Toxic Dimethylarsinous Acid by Rats and Rat Liver Cytosol

Dimethylarsinic acid (DMAs(V)), the major urinary metabolite of inorganic arsenic, is weakly cytotoxic, whereas its reduced form, dimethylarsinous acid (DMAs(III)), is highly toxic. Although glutathione S-transferase omega 1 (GSTO1) and arsenic methyltransferase have been shown or thought to catalyze DMAs(V) reduction, their role in DMAs(V) reduction in vivo, or in cell extracts is uncertain. Therefore, the reduction of DMAs(V) to DMAs(III) in rats and in rat liver cytosol was studied to better understand its mechanism. To assess DMAs(V) reduction in rats, a novel procedure was devised based on following the accumulation of red blood cell (RBC)-bound dimethylarsenic (DMAs), which represents DMAs(III), in the blood of DMAs(V)-injected anesthetized rats. These studies indicated that rats reduced DMAs(V) to DMAs(III) to a significant extent, as in 90 min 31% of the injected 50 μmol/kg DMAs(V) dose was converted to DMAs(III) that was sequestered by the circulating erythrocytes. Pretreatment of rats with glutathione (GSH) depletors (phorone or BSO) delayed the elimination of DMAs(V) and the accumulation of RBC-bound DMAs, whereas the indirect methyltransferase inhibitor periodate-oxidized adenosine was without effect. Assessment of DMAs(V)-reducing activity of rat liver cytosol revealed that reduction of DMAs(V) required cytosolic protein and GSH and was inhibited by thiol reagents, GSSG and dehydroascorbate. Although thioredoxin reductase (TRR) inhibitors (aurothioglucose and Sb(III)) inhibited cytosolic DMAs(V) reduction, recombinant rat TRR plus NADPH, alone or when added to the cytosol, failed to support DMAs(V) reduction. On ultrafiltration of the cytosol through a 3 kDa filter, the reducing activity in the retentate was lost but was largely restored by NADPH. Such experiments also suggested that the reducing enzyme was larger than 100 kDa and was not GSTO1. In summary, reduction of DMAs(V) to the highly toxic DMAs(III) in rats and rat liver cytosol is a GSH-dependent enzymatic process, yet its mechanism remains uncertain.

Glutathione-supported arsenate reduction coupled to arsenolysis catalyzed by ornithine carbamoyl transferase

Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished by ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.

Glutathione synthetase promotes the reduction of arsenate via arsenolysis of glutathione

The environmentally prevalent arsenate (As(V)) undergoes reduction in the body to the much more toxic arsenite (As(III)). Phosphorolytic enzymes and ATP synthase can promote the reduction As(V) by converting it into arsenylated products in which the pentavalent arsenic is more reducible by glutathione (GSH) to As(III) than in inorganic As(V). Glutathione synthetase (GS) can catalyze the arsenolysis of GSH (γ-Glu-Cys-Gly) yielding two arsenylated products, i.e. γ-Glu-Cys-arsenate and ADP-arsenate. Thus, GS may also promote the reduction of As(V) by GSH. This hypothesis was tested with human recombinant GS, a Mg(2+) dependent enzyme. GS markedly increased As(III) formation when incubated with As(V), GSH, Mg(2+) and ADP, but not when GSH, Mg(2+) or ADP were separately omitted. Phosphate, a substrate competitive with As(V) in the arsenolysis of GSH, as well as the products of GSH arsenolysis or their analogs, e.g. glycine and γ-Glu-aminobutyrate, decreased As(V) reduction. Replacement of ADP with ATP or an analog that cannot be phosphorylated or arsenylated abolished As(V) reduction, indicating that GS-supported As(V) reduction requires formation of ADP-arsenate. In the presence of ADP, however, ATP (but not its metabolically inert analog) tripled As(V) reduction because ATP permits GS to remove the arsenolysis inhibitory glycine and γ-Glu-Cys by converting them into GSH. GS failed to promote As(V) reduction when GSH was replaced with ophthalmic acid, a GSH analog substrate of GS containing no SH group (although ophthalmic acid did undergo GS-catalyzed arsenolysis), indicating that the SH group of GSH is important for As(V) reduction. Our findings support the conclusion that GS promotes reduction of As(V) by catalyzing the arsenolysis of GSH, thus producing ADP-arsenate, which upon being released from the enzyme is readily reduced by GSH to As(III).

The mechanism of the polynucleotide phosphorylase-catalyzed arsenolysis of ADP

Using ADP and arsenate (AsV), polynucleotide phosphorylase (PNPase) catalyzes the apparent arsenolysis of ADP to AMP-arsenate and inorganic phosphate, with the former hydrolyzing rapidly into AMP and AsV. However, in the presence of glutathione, AMP-arsenate may also undergo reductive decomposition, yielding AMP and arsenite (AsIII). In order to clarify the mechanism of ADP arsenolysis mediated by Escherichia coli PNPase, we analyzed the time course of the reaction in the presence of increasing concentrations of ADP, with or without polyadenylate (poly-A) supplementation. These studies revealed that increasing supply of ADP enhanced the consumption of ADP but inhibited the production of both AMP and AsIII. Formation of these products was amplified by adding trace amount of poly-A. Furthermore, AMP and AsIII production accelerated with time, whereas ADP consumption slowed down. These observations collectively suggest that PNPase does not catalyze the arsenolysis of ADP directly (in a single step), but in two separate consecutive steps: the enzyme first converts ADP into poly-A, then it cleaves the newly synthesized poly-A by arsenolysis. It is inferred that one active site of PNPase can catalyze only one of these reactions at a time and that high ADP concentrations favor poly-A synthesis, thereby inhibiting the arsenolysis.

Reduction of the Pentavalent Arsenical Dimethylarsinic Acid and the GSTO1 Substrate S-(4-Nitrophenacyl)glutathione by Rat Liver Cytosol: Analyzing the Role of GSTO1 in Arsenic Reduction

Dimethylarsinic acid (DMAs(V)) is the major urinary metabolite of inorganic arsenic. The relatively atoxic DMAs(V) is reduced in the body to the much more toxic and thiol-reactive dimethylarsinous acid (DMAs(III)). Glutathione S-transferase omega 1 (GSTO1) can catalyze this toxification step; however, its role in the reduction of DMAs(V) in vivo or by tissue extracts is unclear. Therefore, we assessed the role of GSTO1 in the reduction of DMAs(V) to DMAs(III) by rat liver cytosol. The experiments revealed that glutathione (GSH) supported the cytosolic DMAs(V) reduction specifically and that GSH analogues and GSH conjugates, such as S-alkylglutathiones and S-(4-nitrophenacyl)glutathione (4-NPG; a GSTO1 specific substrate), inhibited the formation of DMAs(III). Observations in line with the view that GSTO1 catalyzes the cytosolic reduction of DMAs(V) include (i) findings pointing to the presence of a GSH-binding site on the DMAs(V)-reducing cytosolic enzyme, (ii) identical responsiveness of the DMAs(V)- and 4-NPG-reducing activities in rat liver cytosol to the GSTO1 specific inhibitors KT53 and chloromethylfluorescein diacetate, and (iii) perfect coelution of the two activities during affinity and anion exchange chromatography of cytosolic proteins. Other observations appear ambiguous as to the role of GSTO1 in the cytosolic reduction of DMAs(V). These include the different sensitivities of the DMAs(V)-reducing and GSTO1 activities to aurothioglucose, trivalent antimony, and zinc ions, as well as the preserved GSTO1 activity in cytosols whose DMAs(V)-reducing activity was lost due to spontaneous thiol oxidation. These disparate findings may be reconciled by assuming that GSTO1 catalyzes the reduction of both DMAs(V) and 4-NPG in rat liver cytosol; however, the enzyme employs different sites and/or mechanisms when reducing these substrates.

Complex Formation of Resorufin and Resazurin with Β-Cyclodextrins: Can Cyclodextrins Interfere with a Resazurin Cell Viability Assay?

Resazurin (or Alamar Blue) is a poorly fluorescent dye. During the cellular reduction of resazurin, its highly fluorescent product resorufin is formed. Resazurin assay is a commonly applied method to investigate viability of bacterial and mammalian cells. In this study, the interaction of resazurin and resorufin with β-cyclodextrins was investigated employing spectroscopic and molecular modeling studies. Furthermore, the influence of β-cyclodextrins on resazurin-based cell viability assay was also tested. Both resazurin and resorufin form stable complexes with the examined β-cyclodextrins (2.0–3.1 × 103 and 1.3–1.8 × 103 L/mol were determined as binding constants, respectively). Cells were incubated for 30 and 120 min and treated with resazurin and/or β-cyclodextrins. Our results suggest that cyclodextrins are able to interfere with the resazurin-based cell viability assay that presumably results from the following mechanisms: (1) inhibition of the cellular uptake of resazurin and (2) enhancement of the fluorescence signal of the formed resorufin.

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants.

The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme.