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Inhibition of Papain by S-Nitrosothiols

2000; Elsevier BV; Volume: 275; Issue: 27 Linguagem: Inglês

10.1074/jbc.m001054200

1083-351X

Ming Xian, Xinchao Chen, Ziye Liu, Kun Wang, Peng George Wang,

Sulfur Compounds in Biology

S-Nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by NO. The cysteine protease papain has a critical thiol residue (Cys25). It has been demonstrated that NO or NO donors such as sodium nitroprusside and N-nitrosoaniline derivatives can reversibly inhibit this enzyme by S–NO bond formation in its active site. In this study, a different regulated mechanism of inactivation was reported using S-nitrosothiols as the NO donor. FiveS-nitroso compounds,S-nitroso-N-acetyl-dl-penicillamine,S-nitrosoglutathione, S-nitrosocaptopril, glucose-S-nitroso-N-acetyl-dl-penicillamine-2, and the S-nitroso tripeptide acetyl-Phe-Gly-S-nitrosopenicillamine, exhibited different inhibitory activities toward the enzyme in a time- and concentration-dependent manner with second-order rate constants (k i/K I) ranging from 8.9 to 17.2 m−1s−1. The inhibition of papain byS-nitrosothiol was rapidly reversed by dithiothreitol, but not by ascorbate, which could reverse the inhibition of papain by NOBF4. Incubation of the enzyme with a fluorescentS-nitroso probe (S-nitroso-5-dimethylaminonaphthalene-1-sulfonyl) resulted in the appearance of fluorescence of the protein, indicating the formation of a thiol adduct. Moreover, S-transnitrosylation in the incubation of S-nitroso inactivators with papain was excluded. These results suggest that inactivation of papain byS-nitrosothiols is due to a direct attack of the highly reactive thiolate (Cys25) in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between the inactivator and papain. S-Nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by NO. The cysteine protease papain has a critical thiol residue (Cys25). It has been demonstrated that NO or NO donors such as sodium nitroprusside and N-nitrosoaniline derivatives can reversibly inhibit this enzyme by S–NO bond formation in its active site. In this study, a different regulated mechanism of inactivation was reported using S-nitrosothiols as the NO donor. FiveS-nitroso compounds,S-nitroso-N-acetyl-dl-penicillamine,S-nitrosoglutathione, S-nitrosocaptopril, glucose-S-nitroso-N-acetyl-dl-penicillamine-2, and the S-nitroso tripeptide acetyl-Phe-Gly-S-nitrosopenicillamine, exhibited different inhibitory activities toward the enzyme in a time- and concentration-dependent manner with second-order rate constants (k i/K I) ranging from 8.9 to 17.2 m−1s−1. The inhibition of papain byS-nitrosothiol was rapidly reversed by dithiothreitol, but not by ascorbate, which could reverse the inhibition of papain by NOBF4. Incubation of the enzyme with a fluorescentS-nitroso probe (S-nitroso-5-dimethylaminonaphthalene-1-sulfonyl) resulted in the appearance of fluorescence of the protein, indicating the formation of a thiol adduct. Moreover, S-transnitrosylation in the incubation of S-nitroso inactivators with papain was excluded. These results suggest that inactivation of papain byS-nitrosothiols is due to a direct attack of the highly reactive thiolate (Cys25) in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between the inactivator and papain. S-nitrosoglutathione S-nitroso-N-acetyl-dl-penicillamine 5-dimethylaminonaphthalene-1-sulfonyl dithiothreitol 5,5′-dithiobis(2-nitrobenzoate) NO is a newly discovered biological messenger that plays important roles in physiological and pathophysiological conditions such as septic shock, inflammation, and endothelium-dependent vasorelaxation (1.Ignarro L. Murad F. Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications. Academic Press, Inc., San Diego, CA1995Google Scholar, 2.Lancaster J. Nitric Oxide: Principles and Actions. Academic Press, Inc., San Diego, CA1996Google Scholar). Many effects of NO are based on its reaction with other important species. NO reacts with superoxide to make peroxynitrite (3.Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722PubMed Google Scholar). It also forms metal-nitrosyl complexes in the reaction of transition metals (4.Richter-Addo G.B. Acc. Chem. Res. 1999; 32: 529-536Crossref Scopus (60) Google Scholar). The interactions of NO with sulfhydryl-containing molecules and enzymes have gained considerable importance (5.Broillet M.-C. Cell. Mol. Life Sci. 1999; 55: 1036-1042Crossref PubMed Scopus (176) Google Scholar, 6.Gaston B. Biochim. Biophys. Acta. 1999; 1411: 323-333Crossref PubMed Scopus (234) Google Scholar). In many biological systems,S-nitrosylation reactions, transferring NO from a NO donor to a protein sulfhydryl group, affect protein function. Targets for this type of modification, among others, are serum albumin (7.Stamler J.S. Simon D.I. Osborn J.A. Mullins M.E. Jaraki O. Michel T. Singel D.J. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 444-448Crossref PubMed Scopus (1305) Google Scholar), tissue-type plasminogen activator (8.Stamler J.S. Simon D.I. Jaraki O. Osborn J.A. Francis S. Mullins M.E. Singel D.J. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8087-8091Crossref PubMed Google Scholar), transcriptional activators (9.Henderson S.A. Lee P.H. Aeberhard E.E. Adams J.W. Ignarro L.J. Murphy W.J. Sherman M.P. J. Biol. Chem. 1994; 269: 25239-25242Abstract Full Text PDF PubMed Google Scholar), gyceraldehyde-3-phosphate dehydrogenase (10.Mohr S. Stamler J.S. Brune B. FEBS Lett. 1994; 348: 223-227Crossref PubMed Scopus (197) Google Scholar), human immunodeficiency virus protease (11.Sehajpal P.K. Basu A. Ogiste J.S. Lander H.M. Biochemistry. 1999; 38: 13407-13413Crossref PubMed Scopus (26) Google Scholar), and protein-phosphotyrosine phosphatase (12.Caselli 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). Some cysteine proteases such as caspase-3 (13.Rossig L. Fichtlscherer B. Breitschopf K. Haendeler J. Zeiher A.M. Mulsch A. Dimmeler S. J. Biol. Chem. 1999; 274: 6823-6826Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) and cathepsin K (14.Percival M.D. Ouellet M. Campagnolo C. Claveau D. Li C. Biochemistry. 1999; 38: 13574-13583Crossref PubMed Scopus (125) Google Scholar) have also been demonstrated to be inhibited by NO donors. Cysteine proteases (EC 3.4.22.1–18) compose a large class of enzymes from plant, animal, and bacterial sources. They play important roles in various biological processes (for review, see Refs. 15.Lowe G. Tetrahedron. 1976; 32: 291-302Crossref Scopus (140) Google Scholar and 61.Rawlings N.J. Barrett A.J. Methods Enzymol. 1994; 244: 461-486Crossref PubMed Scopus (336) Google Scholar). Since many disease states such as muscular dystrophy, inflammation, and rheumatoid arthritis are associated with elevated proteolytic activity of cysteine proteases, much attention has been paid to the rational design and synthesis of selective inhibitors of these types of enzymes (16.Fischer, G. (1988) Nat. Prod. Rep. 465–495Google Scholar, 17.Otto H. Schirmeister T. Chem. Rev. 1997; 97: 133-171Crossref PubMed Scopus (696) Google Scholar, 18.Babine R.E. Bender S.L. Chem. Rev. 1997; 97: 1359-1472Crossref PubMed Scopus (912) Google Scholar). The active sites of cysteine proteases contain an essential cysteine sulfhydryl and histidine imidazole unit (19.Keillor J.W. Brown R.S. J. Am. Chem. Soc. 1992; 114: 7983-7989Crossref Scopus (39) Google Scholar). The active thiol is highly sensitive to oxidation, which changes the enzyme's properties and sensitivity to inhibitors and activators. It has been suggested that enzyme oxidation occurs in vivo and may represent an important mode of post-translational regulation of enzyme activity (20.Hampton M.B. Fadeel B. Orrenius S. Ann. N. Y. Acad. Sci. 1998; 854: 328-335Crossref PubMed Scopus (237) Google Scholar). Papain, the most studied plant cysteine protease, shares many features with physiologically important mammalian cysteine proteases such as cathepsins B, H, L, and S and the calpains. The x-ray structure for human cathepsin B and earlier comparisons of the crystal structure of papain with models of cathepsins B and H demonstrated that the mammalian enzymes show nearly identical folding patterns to papain, especially around the active site (21.Meara J.P. Rich D.H. J. Med. Chem. 1996; 39: 3357-3366Crossref PubMed Scopus (70) Google Scholar). In the active site of papain, Cys25 and His159 are thought to be catalytically active as a thiolate-imidazolium ion pair. This enzyme should be susceptible to NO donors in a manner similar to other cysteine-containing enzymes. In fact, our previous reports (22.Guo Z. Ramirez J. Li J. Wang P.G. J. Am. Chem. Soc. 1998; 120: 3726-3734Crossref Scopus (29) Google Scholar, 23.Guo Z. McGill A., Yu, L. Li J. Ramirez J. Wang P.G. Bioorg. Med. Chem. Lett. 1996; 6: 573-578Crossref Scopus (16) Google Scholar) have shown that papain can be efficiently inhibited by peptidyl or non-peptidyl N-nitrosoanilines (a novel class of stable NO donors). We demonstrated that the inactivation is due to the formation of a stable S–NO bond in the active site of papain (S-nitroso-Cys25). Other authors, using (E)-ethyl-2-((E)-hydroxyimino)-5-nitro-3-hexenamide and sodium nitroprusside as NO donors to inactivate papain, obtained the same results (24.Venturini G. Fioravanti E. Colasanti M. Persichini T. Ascenzi P. Biochem. Mol. Biol. Int. 1998; 46: 425-428PubMed Google Scholar). S-Nitrosothiols are other important widely used NO donors. These compounds such as GSNO1 may be the most relevant biological molecules to carry out nitrosation reactions under physiological conditions (25.Arnelle D.R. Stamler J.S. Arch. Biochem. Biophys. 1995; 318: 279-285Crossref PubMed Scopus (541) Google Scholar, 26.Padgett C.M. Whorton A.R. Am. J. Physiol. 1995; 269: C739-C749Crossref PubMed Google Scholar). Some authors have even suggested that the actions of the endothelium-derived relaxing factor more closely resemble a low molecular weight nitrosothiol rather than the NO radical itself (27.Myers P.R. Minor Jr., R.L. Guerra Jr., R. Bates J.N. Harrison D.G. Nature. 1990; 345: 161-163Crossref PubMed Scopus (828) Google Scholar). In this study, we investigated the influence ofS-nitrosothiols on papain. Our results show thatS-nitrosothiols are efficient papain inhibitors and work through the formation of mixed disulfide species in the active-site cysteine (Cys25) of the enzyme. This mechanism is different from previous reports using other NO donors, including (E)-ethyl-2-((E)-hydroxyimino)-5-nitro-3-hexenamide, sodium nitroprusside, and N-nitroso compounds (22.Guo Z. Ramirez J. Li J. Wang P.G. J. Am. Chem. Soc. 1998; 120: 3726-3734Crossref Scopus (29) Google Scholar, 23.Guo Z. McGill A., Yu, L. Li J. Ramirez J. Wang P.G. Bioorg. Med. Chem. Lett. 1996; 6: 573-578Crossref Scopus (16) Google Scholar, 24.Venturini G. Fioravanti E. Colasanti M. Persichini T. Ascenzi P. Biochem. Mol. Biol. Int. 1998; 46: 425-428PubMed Google Scholar). Thus, the possibility of another cysteine modification besidesS-nitrosylation should be considered when the effects of S-nitrosothiols on critical thiol-containing proteins are investigated. Enzyme, amino acids, amino acid derivatives, and all other chemicals, solvents, and reagents were obtained from commercial sources (Sigma, Aldrich, and Fluka). They were of the highest purity available and used without further purification unless otherwise noted.1H and 13C NMR spectra were recorded on a Varian VRX 400S NMR apparatus. Silica Gel F254 plates (Merck) and Silica Gel 60 (70–230 mesh; Merck) were used in analytical TLC and column chromatography, respectively. SNAP (1), GSNO (2), andS-nitrosocaptopril (3) were prepared by the methods of Field et al. (28.Field, L., Dilts, R. V., Ravichandran, R., Lenhert, P. G., and Carnahan, G. (1978 ) J. Chem. Soc. Chem. Commun. 249–250Google Scholar), Hart (29.Hart, T. W. (1985) Tetrahedron Lett. 2013–2015Google Scholar), and Loscalzoet al. (30.Loscalzo J. Smick D. Andon N. Cooke J. J. Pharmacol. Exp. Ther. 1989; 249: 726-729PubMed Google Scholar), respectively. Glucose-SNAP-2 (4) was synthesized according to our previous method (31.Ramirez J., Yu, L. Li J. Braunschweiger P.G. Wang P.G. Bioorg. Med. Chem. Lett. 1996; 6: 2575-2580Crossref Scopus (46) Google Scholar). Acetyl-Phe-Gly-OH (0.4 mmol) was reacted with 2-N-acetyl-3-S-triphenylmethyl-3-methylbutanoic acid methyl ester (1 eq) in 10 ml of dimethylformamide at 50 °C overnight with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.5 eq), triethylamine (3 eq), and hydroxybenzotriazole (3 eq) as coupling reagents. The tripeptide was obtained in 30% yield. The free sulfhydryl compound was afforded in 70% yield by deprotection of the triphenylmethyl group with trifluoroacetic acid (0.5 ml) in CH2Cl2. The target compound 5 was obtained in 85% yield by nitrosation of the free sulfhydryl group with ethyl nitrite (4–5 eq) in MeOH/H2O at −5–0 °C for 1.5 h. Compound 5: 1H NMR (CD3OD) δ 7.205–7.295 (m, 5H), 4.613 (d, 2H,J = 6.4 Hz), 3.793–3.968 (m, 2H), 3.722 (s, 3H), 3.161–3.21 (m, 1H), 2.86–2.92 (m, 1H), 1.89 (s, 3H), 1.45 (s, 3H), and 1.394 (s, 3H); 13C NMR (CD3OD) δ 173.2, 172.2, 170.3, 170.1, 137.4, 129.0, 128.3, 126.6, 83.6, 61.7, 55.2, 51.4, 42.4, 37.5, 29.2, 28.8, and 21.2. 5-N-Dansylpentanol (540 mg) was obtained in 95% yield by overnight coupling of dansyl chloride with 1.1 eq of 5-aminopentanol in 40 ml of tetrahydrofuran with triethylamine (3 eq) as a base. Then, the free hydroxyl compound (650 mg) was transferred to tosylate compound in 85% yield with tosyl chloride (1.2 eq) in 50 ml of CH2Cl2/pyridine (1:2.3). The tosylate (1 mmol, 490 mg) was substituted by thiol acetate in 30 ml of acetone quantitatively with potassium thiol acetate. Thus, the free thiol compound was obtained in 60% yield by deprotection of the acetyl group (1 mmol, 395 mg) with diisobutyl aluminum hydride (2.3 eq) in 30 ml of CH2Cl2 at −78 °C for 2.5 h. Compound 6 was obtained in 90% yield by nitrosation of the thiol compound (0.3 mmol) with NaNO2/HCl (1.3 eq) in MeOH/H2O at 0 °C for 2 h. Compound6: 1H NMR (CD3OD) δ 8.92 (d, 1H,J = 8.4 Hz), 8.64 (d, 1H, J = 9.0 Hz), 8.55 (dd, 1H, J = 7.6, 1.2 Hz), 8.16 (d, 1H,J = 7.6 Hz), 7.85–7.91 (m, 2H), 3.51 (s, 6H), 3.30–3.45 (m, 2H), 2.86 (t, 2H, J = 6.6Hz), 1.29–1.36 (m, 4H), and 1.10–1.28 (m, 2H); 13C NMR (CD3OD) δ 139.5, 137.8, 130.0, 129.5, 127.5, 127.4, 126.9, 126.1, 125.5, 119.5, 46.7, 42.4, 38.0, 28.3, 28.2, and 25.4. The solutions of S-nitrosothiols were prepared fresh each time before use. SNAP and S-nitrosodansyl were dissolved in Me2SO and then diluted in reaction buffer to achieve the desired concentration. Twice-crystallized papain was reductively activated at a concentration of 3 mg/ml by incubation with 10 mmdithiothreitol in 50 mm phosphate buffer, pH 6.2, for 1.5 h. It was then passed through a 2.3 × 26-cm Sephadex G-25 column (Amicon, Inc., Danvers, MA) equipped with a Retriever II fraction collector and an ISCO UA-5 absorbance/fluorescence detector at 4–6 °C. Fractions containing the protein were pooled and lyophilized. The protein was stored at −20 °C before use. The enzyme obtained this way excluded any unexpected effect of DTT. Papain activity was determined spectrophotometrically at 410 nm with a Hewlett-Packard 8453 UV/visible spectrophotometer using the chromogenic substrate N-benzyloxycarbonyl-Glyp-nitrophenyl ester (25 mm, 10 μl) in 1 ml of 50 mm phosphate buffer, pH 6.2, 1 mm EDTA, 0.1m NaCl, and 7% (v/v) acetonitrile. A 5 mmnitrosothiol solution (freshly prepared) was prepared in 50 mm phosphate buffer, pH 6.2, 1 mm EDTA, and 0.1m NaCl. To initiate incubation, each nitrosothiol solution obtained after serial dilution was mixed with an equal volume of active papain at 25 °C. An aliquot was periodically removed from the incubation mixture and diluted into the enzyme assay solution containing the substrate. The residual enzyme activity was measured. A control preincubation solution containing all of the ingredients except for the inhibitor itself was run and assayed in parallel. These experiments were performed both in the absence and presence of Gly-Gly-Tyr-Arg. The free thiol group of the protein was titrated by 5-(octyldithio)-2-nitrobenzoate as described by Faulstich et al. (32.Faulstich H. Tews P. Heintz D. Anal. Biochem. 1993; 208: 357-362Crossref PubMed Scopus (37) Google Scholar). This reagent reacts with free thiol on protein faster than Ellman's reagent due to its lipophilic hydrocarbon chain and the absence of one negative charge, which permits its access to thiol groups that normally remain undetected by Ellman's reagent. Free thiols were titrated both before and after the inactivation of the enzyme by inactivators. Titration was performed in the Hewlett-Packard 8453 UV/visible spectrophotometer by measuring the release of 5-thio-2-nitrobenzoate anion (ε412 = 13600m−1cm−1). The free thiol of active papain (1 mg/ml) was titrated by 5-(octyldithio)-2-nitrobenzoate (1.2 mm) in 50 mm phosphate buffer, pH 6.2, 1 mm EDTA, and 0.1 m NaCl. After incubation of the active enzyme (1 mg/ml) with S-nitroso inhibitor (0.5 mm) for 30 min, the protein was purified and concentrated. The free thiol of this inactive papain (1 mg/ml) was then titrated by 5-(octyldithio)-2-nitrobenzoate (1.2 mm). Protein content was determined according to the method of Lowryet al. (33.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Active papain (0.5 mg/ml, 500 μl) was incubated with 500 μl of S-nitroso compounds (1 mm) or nitrosonium tetrafluoroborate (NOBF4; 6 mm) in 50 mm phosphate buffer, pH 6.2, 1 mm EDTA, and 0.1m NaCl at 25 °C for 60 min. The enzyme activity was totally abolished. The protein was then passed through a Sephadex G-25 column for purification. Fractions containing the protein were concentrated by centrifugal filtration (Centricon YM-3, Amicon, Inc.). The obtained inactive enzyme (0.5 mg/ml, 100 μl) was then incubated with 100 μl of l-ascorbic acid (20 mm) or DTT (10 mm), and the activity of papain was measured at different time intervals. Samples without inactivators served as controls. Active papain (1 mg/ml, 600 μl) was incubated with compound6 (2 mm, 600 μl) in 50 mmphosphate buffer, pH 6.2, 1 mm EDTA, and 0.1 mNaCl at 25 °C until the papain was completely inactivated (∼30 min). The protein was then passed through a Sephadex G-25 column to remove excess compound 6 and other low molecular weight reaction products. Fractions containing the protein (5 ml) were concentrated by centrifugation at 7000 rpm at 4 °C to produce a 1-ml final solution. The fluorescence of the protein solution (0.5 mg/ml) was measured using a SPEX Fluro Max spectrometer. A control experiment including all the steps except for incubating papain withN-ethylmaleimide (0.1 mm) for 30 min before adding compound 6 was run and assayed in parallel. Freshly prepared solutions of S-nitroso compounds1–5 (0.4 mm, 0.5 ml) and DTNB (0.6 mm, 0.2 ml) were mixed in a UV quartz cell. Then, 0.5 ml of active papain (0.6 mg/ml) was added to the solution, and the mixture was monitored at 412 nm for 30 min with the Hewlett-Packard 8453 UV/visible spectrophotometer. The reaction was carried out in 50 mm phosphate buffer, pH 6.2, 1 mm EDTA, and 0.1m NaCl at 25 °C. The procedure is based on the low reactivity of DTNB with protein thiol compared with most simple thiols (34.Zhang H. Means G.E. Anal. Biochem. 1996; 237: 141-144Crossref PubMed Scopus (39) Google Scholar). If the reaction of papain with S-nitroso inhibitor wasS-transnitrosation, the generated simple thiol would rapidly react with DTNB to give 5-thio-2-nitrobenzoate dianion. The strong visible absorbance of 5-thio-2-nitrobenzoate dianion (ε412 = 13600 m−1cm−1) could be easily detected. The inhibition of papain byS-nitrosothiol compounds 1-5 was first investigated. SNAP (1), GSNO (2), andS-nitrosocaptopril (4) were used in our bioassay because these three compounds are the most typical and stableS-nitrosothiols and have been widely used in the biological research on nitric oxide. Glucose-SNAP-2 (3) represents a novel class of NO donor compounds developed by our group (31.Ramirez J., Yu, L. Li J. Braunschweiger P.G. Wang P.G. Bioorg. Med. Chem. Lett. 1996; 6: 2575-2580Crossref Scopus (46) Google Scholar). The sugar unit of these compounds enhances water solubility, cell penetration, and drug-receptor interaction and influences the dose-response relationships. Moreover, these compounds are more stable than SNAP in aqueous solution. Compound 5 was specifically designed and synthesized for papain because this enzyme prefers to have a peptide substrate with a bulky residue such as Phe at the S2 subsite and a small hydrophobic residue at the S1 subsite (35.Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4766) Google Scholar, 36.Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1968; 32: 898-902Crossref PubMed Scopus (340) Google Scholar). The inactivation process could therefore be facilitated by directing the nitrosothiol moiety into the active site through the binding of P1 and P2residues to S1 and S2 subsites. The molecular structures of these inactivators are shown in Fig.1. Incubation of papain with each of these S-nitrosothiols resulted in a time- and concentration-dependent loss of enzyme activity. A typical enzyme inactivation process by GSNO is shown in Fig. 2. As shown, the inactivation reactions obeyed apparent first-order kinetics. The apparent first-order inactivation constants (k obs) could be calculated by plotting the residual activity versus time and fitting a linear equation. The replots ofk obs as a function of S-nitrosothiol concentration gave a Kitz-Wilson plot (37.Kitz R. Wilson I.B. J. Biol. Chem. 1962; 237: 3245-3250Abstract Full Text PDF PubMed Google Scholar) (Fig.3), indicating that the inactivation of papain by S-nitrosothiols is a bimolecular process. The calculated kinetic parameters for each inhibitor inactivation of the enzyme are shown in Table I.Figure 3Kitz-Wilson plot for the inhibition of papain by GSNO.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IKinetic parameters for the inactivation of papain by S-nitrosothiolsS-Nitroso compoundsk iK Ik i/K Imin −1mmm −1 s −110.3180.35614.920.2020.30511.130.2390.4498.940.4770.61812.950.0910.09617.2 Open table in a new tab The kinetic results presented in Table I indicated thatS-nitroso compounds were moderate inactivators of papain. The second-order rate constants (k i/K I) for inactivation of papain by these compounds ranged from 17.2 to 8.9m−1 s−1. The values were substantially lower than those exhibited by other well known papain inhibitors such as peptidyl diazomethanes. This result was partly attributable to the higher K I values of theS-nitrosothiols, whereas in the diazomethane case, the actual dissociation constant for the Michaelis-type complex was significantly lower due to the fast reversible chemical step preceding the alkylation step. The intrinsic reactivity of the nitrosothiol moiety toward the papain differed in these five compounds, of which the most potent was inhibitor 5, as we predicted. This result suggested that the Phe and Gly residues in inhibitor 5contributed significantly to the stabilization of the inactivator-enzyme complex. However, by comparing the second-order rate constants for inhibition of papain by S-nitrosothiols with those exhibited by peptidyl N-nitrosoanilines (22.Guo Z. Ramirez J. Li J. Wang P.G. J. Am. Chem. Soc. 1998; 120: 3726-3734Crossref Scopus (29) Google Scholar), we can conclude that the functional S–NO residue is generally a more potent inactivator than the N–NO residue. By comparing the kinetic parameters of SNAP and glucose-SNAP-2, it appeared that the introduction of a sugar fragment decreased the inhibitory potency of SNAP. The same effect was observed for protein-tyrosine phosphatase (38.Xian M. Wang K. Chen X. Hou Y. McGill A. Chen X. Zhou B. Zhang Z.-Y. Cheng J.-P. Wang P.G. Biochem. Biophys. Res. Commun. 2000; 268: 310-314Crossref PubMed Scopus (34) Google Scholar). The next step was to identify the site implicated in the enzyme inactivation. Gly-Gly-Tyr-Arg is a competitive peptide inhibitor of papain. To determine whether the inhibitory effect of S-nitrosothiols on papain occurs due to modification at a similar or distinct site of action, we analyzed the inactivation of papain byS-nitrosothiol in the presence or absence of Gly-Gly-Tyr-Arg. The effects of Gly-Gly-Tyr-Arg alone and together with GSNO are shown in Fig. 4. A combination of GSNO and the tetrapeptide inhibited the enzyme by 42%, whereas individually, 75 and 96% inhibition was achieved, respectively. This result that the competitive inhibitor protected the enzyme from inactivation suggested that the action of S-nitrosothiols was directed to the active site of papain. Since papain has only one free sulfhydryl group (Cys25 in the active site of the enzyme), the data in Fig. 4 also suggested that inactivation of papain by S-nitroso compounds was due to the modification of Cys25 of the protein. To further verify this conclusion, free thiol groups were titrated both before and after inactivation of the enzyme with S-nitroso compounds (data not shown). We found that only <0.05 free thiol groups/enzyme molecule were titrated after incubating the enzyme (1 mg/ml) with S-nitrosothiols (0.5 mm) for 30 min, whereas 0.81 free thiol groups were titrated in the uninhibited enzyme. These results also indicated that the sulfhydryl group in the active site of papain (Cys25) was involved in the enzyme inactivation and that the modification of this sulfhydryl group by inactivators was complete. It is well known that the activities of many cysteine-containing enzymes are modulated by an NO-induced mechanism (39.Stamler J.S. Cell. 1996; 78: 931-936Abstract Full Text PDF Scopus (1633) Google Scholar). The present dogma for the mechanism of inactivation of critical thiol-containing enzymes by NO or NO donors is the formation of an S-nitroso adduct (40.Stamler J.S. Curr. Top. Microbiol. Immunol. 1995; 196: 19-36Crossref PubMed Scopus (197) Google Scholar, 41.Kroncke K.D. Fehsel K. Kolb-Bachofen V. Nitric Oxide. 1997; 1: 107-120Crossref PubMed Scopus (469) Google Scholar). This species, which has been identified by spectroscopic and colorimetric quantitation techniques in a number of proteins, is reducible to the free thiol by DTT with the recovery of enzyme activity (42.Mohr S. Stamler J.S. Brune B. J. Biol. Chem. 1996; 271: 4209-4214Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). This latter criterion has been used to postulate the presence of an S-nitrosothiol group in recent studies on the effects of NO donors on enzymes, sometimes in the absence of direct evidence (43.Venturini G. Colasanti M. Salvati L. Gradoni L. Ascenzi P. Biochem. Biophys. Res. Commun. 2000; 267: 190-193Crossref PubMed Scopus (38) Google Scholar, 44.So H.S. Park R.K. Kim M.S. Lee S.R. Jung B.H. Chung S.Y. Jun C.D. Chung H.T. Biochem. Biophys. Res. Commun. 1998; 247: 809-813Crossref PubMed Scopus (71) Google Scholar, 45.Li J. Billiar T.R. Talanian R.V. Kim Y.M. Biochem. Biophys. Res. Commun. 1997; 240: 419-424Crossref PubMed Scopus (478) Google Scholar). 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