Inhibition of Glutathione Reductase by Dinitrosyl-Iron-Dithiolate Complex
1997; Elsevier BV; Volume: 272; Issue: 35 Linguagem: Inglês
10.1074/jbc.272.35.21767
ISSN1083-351X
AutoresMatthias Boese, Michael Keese, Katja Becker, Rudi Busse, Alexander Mülsch,
Tópico(s)Redox biology and oxidative stress
ResumoThe biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-l-cysteine and dinitrosyl-iron-di-glutathione in a concentration- and time-dependent manner (IC50 30 and 3 μm, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a Ki of 14 μm, and ak3 of 1.3 × 10−3s−1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg2+ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/orS-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specificallyN-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein. The biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-l-cysteine and dinitrosyl-iron-di-glutathione in a concentration- and time-dependent manner (IC50 30 and 3 μm, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a Ki of 14 μm, and ak3 of 1.3 × 10−3s−1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg2+ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/orS-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specificallyN-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein. Nitrosyl transfer from endogenous nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; DNIC, dinitrosyl-iron complex; GR, glutathione reductase; BSA, bovine serum albumin; DEPC, diethyl pyrocarbonate; DTT, dithiothreitol; GC, guanylyl cyclase. carriers such asS-nitrosoglutathione (1Meyer D.J. Kramer H. Özer N. Coles B. Ketterer B. FEBS Lett. 1994; 345: 177-180Crossref PubMed Scopus (146) Google Scholar) and dinitrosyl-iron complex (DNIC) (2Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) to macromolecular targets is regarded as one major mechanism of biological NO signaling (3Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1642) Google Scholar). Evidence has been provided that endogenous low mass S-nitrosothiols (predominantlyS-nitrosoglutathione) and proteinaceousS-nitrosothiols (S-nitroso-hemoglobin,S-nitroso-serum albumin, and other yet unidentified proteins) exist in human erythrocytes (4Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1493) Google Scholar), plasma (5Stamler J.S. Jaraki O. Osborne J. Simon D.I. Keaney J. Vita J. Singel D. Valeri C.R. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7674-7677Crossref PubMed Scopus (1140) Google Scholar) and bronchial secretion (6Gaston B. Reilly J. Drazen J.M. Fackler J. Ramdev P. Arnelle D. Mullins M.E. Sugarbaker D.J. Chee C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10957-10961Crossref PubMed Scopus (583) Google Scholar). A further NO adduct with GSH, GSNOH, was recently postulated as another transport form of NO (7Hogg N. Singh R.J. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (250) Google Scholar).S-Nitrosation of protein thiols or subsequent reactions such as ADP-ribosylation (8Mohr S. Stamler J.S. Brüne B. J. Biol. Chem. 1996; 271: 4209-4214Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), formation of protein disulfide (9Caselli A. Camici G. Manao G. Moneti G. Pazzagli L. Cappugi G. Ramponi G. J. Biol. Chem. 1994; 269: 24878-24882Abstract Full Text PDF PubMed Google Scholar), and cysteine sulfenic acid (10DeMaster E.G. Quast B.J. Redfern B. Nagasawa H.T. Biochemistry. 1995; 34: 11494-11499Crossref PubMed Scopus (133) Google Scholar) may influence protein function by allosteric mechanisms. Furthermore, reversible S-nitrosation of cell membrane-bound proteins may be involved in transmembraneous NO transport (11McDonald B. Reep B. Lapetina E.G. Molina y Vedia L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11122-11126Crossref PubMed Scopus (31) Google Scholar). A concept has been derived from established chemistry to account for the influence of the redox state of the NO moiety on biological NO transfer reactions. According to this concept nitrosation of nucleophilic targets occurs by attack of nitrosonium (NO+)-like species assumed to be present in “NO carriers” such as N2O3,S-nitrosothiols, and certain iron-nitrosyl complexes (3Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1642) Google Scholar,12Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2467) Google Scholar). Less attention has been paid to the influence of the carrier structure on the interaction of NO with macromolecular targets. However, it is conceivable that the NO carrier will direct the NO moiety specifically to macromolecules recognizing the carrier structure, provided the NO adduct is sufficiently stable and the binding kinetics between the carrier and the macromolecules are rapid enough to outbalance the decomposition of the NO carrier adduct. There is also evidence that NO adducts may exhibit intrinsic bioactivity independent of NO release. Thus, the l-stereoisomer ofS-nitrosocysteine was found to exhibit a significantly higher blood pressure lowering activity compared with thed-isomer, suggesting the existence of stereospecificS-nitroso-l-cysteine receptors in the cardiovascular system (13Travis, M. D. & Lewis, S. J. (1997) Am. J. Physiol. in press.Google Scholar). To assess the influence of the carrier structure on the interaction of NO with a given macromolecule we chose glutathione reductase (GR; EC1.6.4.2) as a model target, and low mass dinitrosyl-iron complexes withl-cysteine and glutathione ligands as NO carriers. These complexes exhibit S-nitrosating activity toward serum albumin in vitro (2Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), and protein-bound forms exist in vivo in animal tissues expressing inducible NO synthase activity (14Chamulitrat W. Jordan S.J. Mason R.P. Litton A.L. Wilson J.G. Wood E.R. Wolberg G. Molina y Vedia L. Arch. Biochem. Biophys. 1995; 316: 30-37Crossref PubMed Scopus (47) Google Scholar). The flavoenzyme GR catalyzes the NADPH dependent reduction of oxidized glutathione (GSSG) to maintain a high intracellular level of GSH. GR carries a redox-active disulfide (Cys-58–Cys-63) in its active site which is reduced by electron transfer from NADPH via the flavin (15Williams Jr., C.H. Müller F. Chemistry and Biochemistry of Flavoenzymes. III. CRC Press, Boca Raton, FL1992: 121-211Google Scholar). Recently it has been shown that GR is inhibited by certain NO carriers (S-nitrosoglutathione, sodium nitroprusside,S-nitroso-N-acetyl-dl-penicillamine) in millimolar concentrations (16Becker K. Gui M. Schirmer R.H. Eur. J. Biochem. 1995; 234: 472-478Crossref PubMed Scopus (79) Google Scholar), suggesting that GR is a potential target for nitrosation reactions. We show here that low mass dinitrosyl-iron complexes in concentrations that may be present under pathophysiological conditions irreversibly inhibit GR, possibly via N-nitrosation. We furthermore demonstrate that the inhibitory potency of the dinitrosyl-iron moiety increases with structural resemblance of the NO carrier to the natural GR substrate, GSSG. GR from bovine intestinal mucosa, fatty acid-free bovine serum albumin (BSA), 2,3-diaminonaphthalene,l-cysteine, glutathione (oxidized and reduced), diethyl pyrocarbonate (DEPC), and Sephadex G-25 were supplied by Sigma, Deisenhofen, Germany. 1-Nitroso-2-hydroxynaphthalene-3,6-disulfonic acid and 1-nitrosopyrrolidine were obtained from Aldrich, Deisenhofen, Germany. NO gas was prepared by reaction of FeSO4 (Fluka, Buchs, Switzerland) with NaNO2 in 5 n HCl and was purified by low temperature high vacuum (p = 0.01 mm Hg) distillation (2Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Paramagnetic dinitrosyl-iron complexes of the type ((NO)2Fe(RS)2) × 18 RSH (RS = l-cysteine, or GSH) were synthesized by mixing evacuated (5 min high vacuum) solutions of FeSO4(5 mg/ml) and neutralized thiols (72 mm) in a Thunberg-type reaction vessel under pure NO gas (PNO 500 mm Hg). NO was added 3 min before mixing. The solution immediately turned dark green and was evacuated after 1 min for a further 2 min to remove excessive NO. The solution bearing ((NO)2Fe(RS)2) in >98% yield with respect to iron was immediately frozen and stored in liquid nitrogen. S-Nitroso-l-cysteine andS-nitroso-BSA were prepared at 4 °C by mixing eitherl-cysteine (100 mm) or fatty acid-free BSA (2 mm) for 10 min with an equimolar amount of sodium nitrite dissolved in 0.5 m H2SO4. TheS-nitrosothiols were frozen and stored in liquid nitrogen. The yield of both S-nitrosothiols was >90% with respect to free thiol added (BSA-thiol/BSA = 0.4 ± 0.04), using molar absorption coefficients of S-nitroso-BSA (ε338= 870 m−1 cm−1, 1) andS-nitroso-l-cysteine (ε547 = 16.7m−1 cm−1) (17Stamler J.S. Osborne J.A. Jaraki O. Rabbani L.E. Mullins M. Singel D.J. Loscalzo J. J. Clin. Invest. 1993; 91: 308-318Crossref PubMed Scopus (839) Google Scholar). At concentrations > 1 μm S-nitrosothiols were assessed by diazotization of sulfanilamide and azocoupling withN,N-ethylendiamine in the presence and absence of Hg2+ ions (3 mm) according to Saville (18Saville B. Analyst. 1958; 83: 670-672Crossref Google Scholar) as described recently (2Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In the nanomolar range nitrite and S-nitrosothiols were quantified by an acid-catalyzed intramolecular diazotization reaction of 2,3-diaminonaphthalene with nitrite forming the highly fluorescent product 2,3-diaminonaphthotriazole (19Misko T.P. Schilling R.I. Salvemini D. Moore W.M. Currie M.G. Anal. Biochem. 1993; 214: 11-16Crossref PubMed Scopus (968) Google Scholar). The sample (570 μl) was mixed with 30 μl of 0.1 m potassium Pibuffer, pH 7.4, and 84 μl of freshly prepared 2,3-diaminonaphthalene (0.05 mg/ml in 1 m HCl). To release NO fromS-nitrosothiols the buffer contained 20 mmHgCl2. Hg2+ did not interfere with the fluorescent assay. After 10 min of incubation at 20 °C in the dark the reaction was terminated by addition of 42 μl of 4.5 mNaOH to maximize the intensity of the fluorescent signal (19Misko T.P. Schilling R.I. Salvemini D. Moore W.M. Currie M.G. Anal. Biochem. 1993; 214: 11-16Crossref PubMed Scopus (968) Google Scholar). The fluorescence was measured with excitation at 375 nm and emission at 415 nm (Deltascan, Photon Technology). Emitted light was detected by a photon-counting photomultiplier (D-104, Photon Technology) and the photomultiplier digital output was collected by an IBM-compatible computer. The content of S-nitrosothiol was calculated by the difference of emission readings of Hg2+-containingversus Hg2+-free samples. A calibration curve was established in each experiment with freshly synthesizedS-nitroso-l-cysteine and sodium nitrite as standards (0.02–2 μm). The detection limit was 20 nm. EPR spectra were recorded on a Bruker EPR 300E spectrometer at 20 °C on solutions (25 μl) filled in a quartz capillary tube (1 mm, inner diameter). The measurements were performed with a modulation amplitude of 1 Gauss, a microwave frequency of 9.6 GHz, a microwave power of 20 mW, and a time constant of 0.2 s. The concentration of dinitrosyl-iron complex was calculated by comparison with the EPR signal of a standard low molecular mass dinitrosyl-iron complex based on double integration of the first derivative EPR signals. Histidine residues were carboxylated by adding a 100-fold molar excess of DEPC and subsequent incubation for 5 min at 20 °C (20Lee M. Arosio P. Cozzi A. Chasteen N.D. Biochemistry. 1994; 33: 3679-3687Crossref PubMed Scopus (118) Google Scholar). Thiol groups were blocked by incubation of the protein (5 μm protein in 0.1m potassium phosphate buffer, pH 7.4) for 5 min at 20 °C with Hg2+ (15 μm) in the presence of NADPH (1 mm). To further demonstrate the involvement of catalytic site thiols in DNIC-induced inhibition of GR the enzyme (5 μm) was preincubated with Hg2+ (15 μm; 5 min at 20 °C) prior to incubation with DNIC-GSH (30 μm; 10 min at 37 °C). Controls were performed without Hg2+ and in the absence and presence of DNIC. The solutions (100 μl) were desalted at 5 °C by passing over a Sephadex G50 Nick® column (Pharmacia) equilibrated with assay buffer (see below) and then incubated with dithiothreitol (DTT) (10 mm, 37 °C) for up to 70 min. After 10, 30 or 70 min of DTT-treatment the activity of GR was assessed in 1:250 diluted aliquots as described below. The reduction of GSSG by GR was determined at 20 °C by monitoring the oxidation of NADPH at 340 nm (ε340 = 6200m−1 cm−1) (21Worthington D.J. Rosemeyer M.A. Eur. J. Biochem. 1976; 67: 231-238Crossref PubMed Scopus (160) Google Scholar). The enzyme was diluted in assay buffer (200 mm potassium chloride, 1 mm EDTA, 50 mm potassium phosphate, pH 6.9). To avoid an interference by NADPH-oxidase activity both reference and sample cuvettes contained NADPH (0.38 mm) and GR (3.5–20 nm) in a final volume of 1 ml. The reaction was started by addition of GSSG (1 mm) to the sample cuvette. The enzyme activity was calculated from the initial rate of the absorbance decrease at 340 nm during 3 min of incubation. For establishment of concentration-response relationships, 1–5 μm GR was preincubated with inhibitors for 30 min and then diluted 300–1000-fold in the final assay mixture. For characterizing individual residues of the bovine GR, the numbering system of the well studied human enzyme (15Williams Jr., C.H. Müller F. Chemistry and Biochemistry of Flavoenzymes. III. CRC Press, Boca Raton, FL1992: 121-211Google Scholar, 16Becker K. Gui M. Schirmer R.H. Eur. J. Biochem. 1995; 234: 472-478Crossref PubMed Scopus (79) Google Scholar) was used; the active site thiols are Cys-58 and Cys-63, and the catalytic imidazole is His-467. Irreversible inhibition kinetics were assessed according to Kitz and Wilson (22Kitz R. Wilson I.B. J. Biol. Chem. 1962; 237: 3245-3249Abstract Full Text PDF PubMed Google Scholar) based on the following reaction scheme (Equation 1).I+E↔KiE⊕I→k3E*→k4E+IEquation 1 If k3, the rate constant for transformation of the reversible enzyme-inhibitor complex (E⊕ I) into the inhibited enzyme (E*), is relatively small, enzyme (E) and inhibitor (I) are in equilibrium with the reversible inhibitor enzyme complex. Also, in case of irreversible inhibition k4 can be neglected. The integrated rate law derived from mass balances readsln[E][E0]=−k31+Ki[I]·tEquation 2 For [I] ≫ [E0],kapp=k31+Ki[I]Equation 3 or1kapp=1k3+Kik3·1[I].Equation 4 k3 is the first order rate constant at high inhibitor concentrations ([I] ≫ Ki). At low inhibitor concentrations the kinetics are in accordance with a simple bimolecular mechanism (kapp = (k3/Ki)·I), and the second order rate constant is (k3/Ki).Ki and k3 were derived from a double-reciprocal plot of the apparent first order rate constantskapp versus concentration of inhibitor [I], i.e. dinitrosyl-iron complex. It should be noted that Ki differs from the half-maximal inhibitory concentration (IC50), sinceKi describes the reversiblepre-equilibrium, while the IC50 refers to the subsequentirreversible reaction. To assess the release of bioactive NO from inhibited GR soluble guanylyl cyclase (GC) purified to apparent homogeneity from bovine lung was used as a detector system (23Mülsch A. Gerzer R. Methods Enzymol. 1991; 195: 377-385Crossref PubMed Scopus (23) Google Scholar). GC activity was measured by the formation of [32P]cGMP from [α-32P]GTP (23Mülsch A. Gerzer R. Methods Enzymol. 1991; 195: 377-385Crossref PubMed Scopus (23) Google Scholar). Inhibited GR- or NO-containing substances (40 μl) were incubated for 10 min at 37 °C in a final volume of 100 μl with GC assay buffer. The final mixture contained triethanolamine-HCl (50 mm, pH 7.4), 3-isobutyl-1-methylxanthine (0.5 mm), [α-32P]GTP (0.2 mm; 0.2 μCi), cGMP (0.1 mm), GSH (2 mm), MgCl2 (3 mm), creatine phosphate (10 mm), creatine phosphokinase (5 units), γ-globulin (0.1 mg/ml), superoxide dismutase (0.3 μm), and 0.8 μg of purified GC. Enzymatic cGMP formation was stopped by addition of zinc acetate (450 μl; 110 mm) and sodium carbonate (450 μl; 120 mm). [32P]cGMP was isolated by chromatography on acid alumina and quantified by liquid scintillation counting. To study the influence of DNIC on the activity of isolated GR, the enzyme was incubated with different concentrations of DNIC-l-cysteine and DNIC-GSH in the presence of NADPH and substrate (GSSG). The GSSG-driven consumption of NADPH was monitored continuously by recording the decrease in absorbance at 340 nm (Fig. 1). During 3 min of reaction at 20 °C, samples containing DNIC-GSH exhibited a nearly constant rate of decrease in absorbance (data not shown), which was inversely related to the concentration of DNIC used. In contrast, NADPH consumption in DNIC-l-cysteine containing reaction mixtures initially exhibited exponential kinetics (Fig. 1, Aand B), indicating that the degree of inhibition of GR by this DNIC derivative progressively increased with time. At longer preincubation periods (>20 min) in the absence of GSSG the kinetics of inhibition by DNIC-l-cysteine became more linear (data not shown). Thus, it is conceivable that during incubation of DNIC-l-cysteine with GR in the presence of GSSG DNIC-GSH was formed by reaction with enzymatically generated GSH. In addition, during preincubation in the absence of GSSG an intrinsic slow inhibitory action of DNIC-l-cysteine was revealed (Fig. 1 B). Therefore, to establish the dose-response relationship for inhibition of GR by DNIC, the enzyme (1 μm) was preincubated at 20 °C in the absence of GSSG with different concentrations of low molecular mass DNIC (6–200 μm) in buffer containing 1 mm NADPH. The enzyme activity was then measured after 30 min of preincubation. It decreased in a DNIC concentration-dependent manner (Fig. 2). The GSH complex was about 10-fold more potent than the l-cysteine complex, although both were equally efficacious. 50% inhibition was elicited by 3 ± 1 μm DNIC-GSH and by 30 ± 3 μmDNIC-l-cysteine. No direct oxidation of NADPH or reduction of NADP+ by DNIC could be observed.Figure 2Inhibition of GR by DNIC-GSH and DNIC-l-cysteine. The enzyme (1 μm) was exposed to different concentrations of DNIC-GSH (•) and DNIC-l-cysteine (○, 6–200 μm) in assay buffer containing 1 mm NADPH at 20 °C. After 30 min, GR activity was determined and related to the activity of the enzyme which was incubated in the absence of the inhibitor (100%). Means ± S.E. (error bars) of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) GR carries a redox-active disulfide (Cys-58–Cys-63) at its catalytic site, which is reduced after binding of NADPH. To investigate whether the inhibition by DNIC depends on the redox state of the enzyme, GR (5 μm) was incubated with DNIC-GSH (30 μm) at 37 °C in the presence and absence of NADPH (1 mm). The activity of GR decreased to 5 ± 2% (n = 4) of control in the presence of NADPH, but did not change in its absence (100 ± 3%, n = 4). This finding suggests that only the reduced enzyme is susceptible to inhibition by DNIC. Since the homodimeric form is essential for catalytic function of GR (15Williams Jr., C.H. Müller F. Chemistry and Biochemistry of Flavoenzymes. III. CRC Press, Boca Raton, FL1992: 121-211Google Scholar) we assessed whether DNIC influenced the aggregation state of the native enzyme by gel permeation chromatography (Superose 200, Pharmacia). DNIC-treated and untreated GR migrated as single peaks at identical positions with an apparent molecular mass of 100 kDa. Therefore DNIC does not inhibit GR by promoting dissociation of the homodimeric enzyme. NO-mediated oxidation and S-nitrosation of protein thiols usually is reversible by an excess of low molecular weight thiols (1Meyer D.J. Kramer H. Özer N. Coles B. Ketterer B. FEBS Lett. 1994; 345: 177-180Crossref PubMed Scopus (146) Google Scholar,24Cleland W.W. Biochemistry. 1964; 3: 480-486Crossref PubMed Scopus (1525) Google Scholar, 25Pietraforte D. Mallozzi C. Scorza G. Minetti M. Biochemistry. 1995; 34: 7177-7185Crossref PubMed Scopus (76) Google Scholar). To test the reversibility of the GR modification by DNIC, the complex-inhibited GR was treated with 5 mm DTT or dimercaptopropanol (30 min, 20 °C); neither reducing agent was able to restore enzyme activity. Moreover, dilution (1000-fold) of the inhibited enzyme in thiol-free or thiol-containing buffer failed to restore activity even after 24 h. To test the accessibility of low mass thiols to the catalytic site thiols in GR, we analyzed the reversibility of Hg2+-elicited inhibition of GR by dithiols. Hg2+ exhibits a high affinity for thiol groups. GR (5 μm) was inhibited by the treatment with Hg2+ (15 μm) within 60 s at 20 °C. DTT and dimercaptopropanol regenerated the initial activity (5 mm, 10 min, 20 °C) of the enzyme, indicating that the Hg2+-bound thiol groups at the active center were accessible to both agents (see also Williams (15Williams Jr., C.H. Müller F. Chemistry and Biochemistry of Flavoenzymes. III. CRC Press, Boca Raton, FL1992: 121-211Google Scholar)). Therefore the dithiols used should be able to interact with the thiol groups in the complex-inhibited enzyme without sterical hindrance. Altogether these findings show that inhibition of GR by DNIC is irreversible. To assess the involvement of cysteine-thiols within the catalytic site in DNIC-induced inhibition of GR we examined whether or not pretreatment of GR by Hg2+, which inhibits GR in a thiol-reversible manner (see above), affords protection against the irreversible inhibition of GR by DNIC-GSH. Since GR contains 3 cysteine thiols per subunit, NADPH-reduced GR was pretreated with a 2-fold molar excess of Hg2+ prior to incubation with a maximally inhibitory concentration of DNIC-GSH (30 μm). Half of the original GR activity was restored within 10 min following incubation of Hg2+/DNIC- and Hg2+- treated GR with DTT. DTT, however, failed to restore catalytic activity to GR treated with DNIC only. The degree of inhibition after 10 min of DTT treatment was: DNIC-treated GR, 85 ± 10%; Hg2+-treated GR, 45 ± 4%; Hg2+/DNIC-treated GR, 52 ± 7% (mean ± S.E.; n = 3). This inhibition was not significantly altered after either 30 or 70 min of treatment with DTT. Thus, Hg2+ pretreatment protects GR from inhibition by DNIC. Consequently, thiols within the catalytic site of GR appear to be the main targets of DNIC and are involved in the irreversible inhibition. To study the kinetics of GR inhibition by DNIC the enzyme (1 μm) was exposed at 20 °C to 0, 6, 10, 15, 20, 35, 50, or 200 μm DNIC-GSH in assay buffer containing 1 mm NADPH. Aliquots were taken and tested for GR activity after different time intervals (0, 2, 6, 12, 18, 24, and 30 min). The time course of inhibition displayed first order kinetics at all DNIC concentrations used. In Fig. 3the natural logarithm of the ratio from the remaining activity (E) and the initial activity (E0) is plotted versus the time of incubation yielding straight lines according to Equations Equation 2, Equation 3, Equation 4 (see “Experimental Procedures”). The slopes represent the rate constants kapp for the inhibition by the corresponding DNIC concentration and were replotted according to Kitz-Wilson (1/kapp versus 1/[I]) as shown in Fig. 4. A linear relationship was apparent which was used to calculate the kinetic constants by linear regression analysis (r = 0.95). The constant for the conversion of the reversible enzyme-inhibitor-complex to the irreversibly inhibited enzyme (k3) amounted to 1.3 × 10−3s−1, the dissociation constant of the reversible complex (Ki) was 14 μm.Figure 4Kitz-Wilson replot of GR inhibition by DNIC-GSH. Double reciprocal plot of kapp(Fig. 3) versus DNIC concentration. Values for 15, 20, and 35 μm DNIC were taken from separate experiments. The dissociation constant Ki of the reversible enzyme-inhibitor complex was derived from the intercept of the graph obtained by linear regression analysis with the horizontal axis. The rate constant for irreversible inhibitionk3 is represented by the reciprocal value of the intercept with vertical axis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The following experiments were performed to reveal the molecular mechanism of inhibition of GR by DNIC. The dinitrosyl-iron moiety of low molecular DNIC binds to free thiol groups of proteins due to a thiol-ligand exchange reaction (2Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 26Mülsch A. Mordvintcev P.I. Vanin A.F. Busse R. FEBS Lett. 1991; 294: 252-256Crossref PubMed Scopus (149) Google Scholar). To assess whether or not the dinitrosyl-iron group was attached to the inhibited protein, GR (5 μm) was incubated with different concentrations of DNIC-GSH or DNIC-lcysteine (5–50 μm) at 20 or 37 °C. After 3, 10, and 60 min the mixture was analyzed by EPR spectroscopy at 20 °C. Recording EPR signals at room temperature allows discrimination between DNIC bound to low and high molecular mass ligands, since the former exhibits an isotropic signal at gav 2.03 with 13-line hyperfine structure (Fig. 5 a), while the latter is characterized by an anisotropic signal at g⊥ 2.04 and g∥ 2.01 (Fig. 5 c). The initial reaction mixtures exhibited exclusively the EPR signal of the low molecular weight DNIC (Fig. 5 b). After 60 min of reaction this signal completely disappeared (Fig. 5 d) because of decomposition of low mass DNIC. The characteristic signal of the protein-bound DNIC (serum albumin-DNIC; Fig. 5 c) was not detectable at any time, though the enzyme was completely inhibited after 30 min of incubation. These findings show that inhibition of GR does not involve formation of a stable DNIC-protein linkage. Since free cysteine thiols within proteins can be nitrosated by low molecular weight DNIC (2Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), we assessed whether this covalent modification accounts for inhibition of GR by DNIC. Therefore DNIC-inactivated enzyme (5 μm, 95 ± 3% inhibited) was passed through a desalting column (Sephadex G-25) to remove excess inhibitor. The protein was concentrated by centrifugation (Ultrafree 30-kDa cut-off, Millipore) and assayed for NOx andS-nitrosothiol (18Saville B. Analyst. 1958; 83: 670-672Crossref Google Scholar). Neither chromatography nor centrifugation reversed inactivation of GR. In the presence of Hg2+ 0.76 ± 0.04 mol nitrite/mol inactive GR was detected (n = 3), but a similar amount of nitrite was found in the absence of Hg2+ (0.75 ± 0.08). This indicates that inhibited GR contains a Griess-reactive NO moiety, which is not bound to a thiol group. Hence it was investigated whether a N- orC-nitrosation of GR by DNIC accounts for inhibition.N-Nitrosopyrrolidine, a nitrosamine of a cyclic secondary amine, also released NO independently of Hg2+ under the acid conditions of the Griess reaction (0.25 m HCl). After 30 min 50 ± 4 μm nitrite was generated by this agent (100 μm). In contrast, the C-nitroso compound 1-nitroso-2-hydroxynaphthalene-3,6-disulfonic acid (50 μm, 60 min incubation), failed to give a positive Griess reaction, either in the presence or absence of Hg2+. To increase the s
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