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Bioconjugate Chem. 2000, 11, 682−687
A Novel Selenocystine-β-Cyclodextrin Conjugate That Acts as a Glutathione Peroxidase Mimic Xiaojun Ren,†,‡ Junqiu Liu,† Guimin Luo,*,† Yan Zhang,† Yuanming Luo,† Ganglin Yan,† and Jiacong Shen‡ Key Laboratory of Molecular Enzymology and Engineering, Jilin University, Changchun 130023, China, and Key Laboratory of Supramolecular Structure & Spectroscopy, Jilin University, Changchun 130023, P. R. China . Received January 28, 2000; Revised Manuscript Received May 1, 2000
A novel artificial glutathione peroxidase mimic consisting of a selenocystine-di-β-cyclodextrin conjugate (selenium-bridged-6,6′-amino-selenocystine-6,6′-deoxy-di-β-cyclodextrin), in which selenocystine is bound to the primary side of β-cyclodextrin through the two amino nitrogen groups of selenocystine, was synthesized. The glutathione peroxidase activities of the mimic-catalyzed reduction of H2O2, tertbutylhydroperoxide, and cumene hydroperoxide by glutathione are 4.1, 2.11, and 5.82 units/µmol, respectively. The first activity was 82 and 4.2 times as much as that of selenocysteine and ebselen, respectively. Studies on the effect of substrate binding on the glutathione peroxidase activity suggest that it is important to consider substrate binding in designing glutathione peroxidase mimics. The detailed steady-state kinetic studies showed that the mimic-catalyzed reduction of H2O2 by glutathione followed a ping-pong mechanism, which was similar to that of the native glutathione peroxidase.
INTRODUCTION
Glutathione peroxidase (GPX)1 (EC 1.11.1.9) comprises four identical subunits. Each subunit approximates 21 000 Da and contains a selenocysteine (SeCys) residue, which is the catalytically active group (1). The crystal structure of the enzyme has been determined by X-ray diffraction method (2). The enzyme catalyzes the reduction of a variety of hydroperoxides. It is believed that many diseases are accompanied or even caused by “oxidative stress”, a situation characterized by excessive formation of reactive reduced oxygen metabolites (superoxide anionic radical, hydrogen peroxide, and hydroxyl radical) (3, 4). To scavenge reactive reduced oxygen, antioxidant pharmacotherapy has emerged as remedy for pathological states characterized by an overwhelmed antioxidant defense system. GPX plays a vital important role in scavenging active oxygen. However, the native GPX has some shortcomings such as instability, poor availability, and high molecular weight, which have limited its application. Therefore, many GPX mimics have been synthesized. They include catalytic antibodies (5, 6), semisynthetic enzyme (7, 8), ebselen (9), diaryl diselenides (10), R-(phenylselenenyl) ketones (11), diaryl ditellurides (12), diaryl tellurides (13), cyclic selenamides (14, 15), and transition metal complex (4). Among them, ebselen is the best-known GPX mimic. This interesting * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: 86-431-8923907. Phone: 86-4318922331-3698. † Key Laboratory of Molecular Enzymology and Engineering. ‡ Key Laboratory of Supramolecular Structure & Spectroscopy. 1 Abbreviations: t-BuOOH, tert-butylhydroperoxide; CuOOH, cumene hydroperoxide; β-CD, β-cyclodextrin; disecysCD, selenium-bridged-6,6′-amino-selenocystine-6,6′-deoxy-β-cyclodextrin; DMF, N,N-dimethylformamide; EDTA, etheylenediaminetetraacetic acid; NADPH, β-nicotinamide adenine dinucleotide phosphate, reduced form; GPX, glutathione peroxidase; GSH, glutathione; PhSeSePh, diphenyl diselenide; SeCys, selenocysteine; SeCyss, selenocystine; TsCl, p-toluene sulfochloride.
molecule has been researched extendedly from pulseradiolytic studies on radical reactivity through its biological properties in organs to clinical settings (16, 17). Enzymes promote very fast reactions by bringing the reacting groups together under the special conditions of the enzyme-substrate complex, but it is clear that a major part of rate enhancement observed is simply due to the way that the functional groups involved are brought together (18). General conclusions from work on models are that efficient catalysis involves an initial binding interaction between the substrate and the enzyme (19). Cyclodextrins are cyclic oligosaccharides consisting of a hydrophobic cavity with which many complexes can be formed via host-guest chemistry. This property has been extensively exploited in the past as enzyme mimics, catalyzing various reactions (20, 21). The effect of an amino group on the antioxidant activity has been extensively investigated with enzyme model (22), but the effect of the substrate binding on the antioxidant activity has not been studied using enzyme model. In this report, β-cyclodextrin (β-CD) modified by selenocystine (SeCyss) was prepared and used as native GPX model to investigate the function of hydrophobic substrate binding. The GPX activities and kinetic studies of the mimic were described. These results reveal that the mimic is a much more efficient catalyst than ebselen, SeCys, and some of the compounds based on diary diselenides. EXPERIMENTAL PROCEDURES
Instrumental. The characterization was performed with a Varian Unity-400 NMR Spectrometer, a Bruker IFS-FT66V Infrared Spectrometer and a Perkin-Elmer 240 DS Elemental Analyzer. The content and valence of selenium were determined by means of an ESCALAB MKII X-ray Photoelectron Spectrometer. The enzyme activities were measured using a Shimadzu UV-3100 Spectrophotometer. Materials. β-CD, p-toluene sulfochloride (TsCl) and tert-butylhydroperoxide (t-BuOOH) were purchased from
10.1021/bc0000076 CCC: $19.00 © 2000 American Chemical Society Published on Web 08/31/2000
Cyclodextrin Mimic of Glutathione Peroxidase
Bioconjugate Chem., Vol. 11, No. 5, 2000 683
Scheme 1
Tianjin Chemical Plant. Sodium borohydride, glutathione (GSH), β-nicotinamide adenine dinucleotide phosphate (NADPH), SeCyss, cumene hydroperoxide (CuOOH), and GSH reductase (type III) were obtained from sigma. Sephadex G-25 was purchased from Pharmacia. Potassium iodide was obtained from Northeast Pharmaceutical Headquarter Factory. β-CD was recrystallized twice from water and dried for 12 h at 120 °C in a vacuum. Analytical grade pyridine was predried by refluxing over KOH for 12 h, then over extremely anhydrous BaO for 12 h, and distilled just before use. N,N-Dimethylformamide (DMF) was dried over P2O5 for several days at room temperature. Then DMF was decanted, shaken with KOH pellets, and then distilled under reduced pressure. All the other materials were used without further purification. Buffers were prepared with distilled, deionized water. Synthesis of Selenium-Bridged-6,6′-Amino-selenocystine-6,6′-deoxy-β-cyclodextrin (disecysCD). The synthesis route of disecysCD is shown in Scheme 1. The regiospecific monotosylation of 6-position hydroxyl of β-CD was carried out according to the ref 23 to synthesize 6-OTs-6-deoxy-β-CD 2. 6-Iodo-6-deoxy-β-CD 3 was synthesized by using the reaction of 6-OTs-6-deoxy-β-CD and potassium iodide in dried DMF at 80 °C (24). The synthesis of disecysCD 4 was accomplished as follows. SeCyss (10 mg, 0.03 mmol) in water solution (4 mL) containing Na2CO3 (13 mg, 0.12 mmol) was added to 6-iodo-6-deoxy-β-CD (400 mg, 0.32 mmol) in DMF (4 mL). The mixture was bubbled using pure nitrogen for 30 min. Under the protection of pure nitrogen, the mixture was kept for 20 h at 65 °C, and then was oxidized in air, finally purified by centrifugation and Sephadex G-25 column (Φ5 × A50 cm) chromatography (λ ) 254) using distilled, deionized water as eluent. The product solution was freeze-dried and the lyophilized powder was fresh yellow product with 27% yield. Characterization of DisecysCD. The structure of disecysCD was analyzed by means of elemental analysis, IR, 1H NMR. The data were shown as follows. Anal. calcd for C90H148O72N2Se2‚6H2O: C, 40.39; H, 6.03; N, 1.05. Found: C, 40.50; H, 5.89; N, 1.06. IR (KBr): 3364 (-OH), 2929, 2854 (CH, CH2), 1671 (CdO), 1615, 1155, 1085, 1029 (-O-). 1H NMR (400 MHz, D2O) d: 5.07-4.82 (m, 14H, 1-H), 3.90-3.59 (m, 54H, 3-, 5-, 6-H, and CH of SeCyss), 3.57-3.33 (m, 28H, 2-, 4-H), 3.30-2.60 (m, 8H, 6-H and CH2 of SeCyss). Determination of the Se Content and the Valence of DisecysCD. The Se content and valence of the mimic were determined by X-ray photoelectron spectroscopy (25). The energy of the exciting X-ray was 1253.6 eV (Mg,
KR). C1s ) 285.0 eV was served as standard. The scans were performed five times. The Se3d electron binding energy of disecysCD was 54.8 eV, which approaches the binding energy 55.1 eV of SeCyss. This result is consistent with our previous observations in 6-SeCD, a GPX mimic containing diselenium bridge (26), indicating that the selenium in disecysCD presents in the form of diselenium bridge (-Se-Se-). The ratio of C/Se and C/N was also given by the experiment as follows: C/Se, 45.9:1 (calcd 45:1), and C/N, 45.5:1 (calculated 45:1), indicating that 1 mol of the mimic contains 2 mol of selenium and nitrogen. Measurement of the binding constant for complexation of GSH by 6-OTs-6-deoxy-β-CD. The binding constant between 6-OTs-6-deoxy-β-CD and GSH in a pH 7.0, 50 mM potassium phosphate buffer was determined by following the absorption at 274 nm of 6-OTs-6-deoxy-β-CD (6.0 × 10-5 M) as a function of the concentration of GSH (1.2 × 10-3 M, 1.8 × 10-3 M, 2.4 × 10-3 M, 6 × 10-3 M) (27). The binding constant was found to be 1.01 × 102 M. Determination of the GPX Activity of DisecysCD. The GPX activity of disecysCD was measured using coupled test procedure assay (10). The reaction was carried out at 37 °C in 500 µL of the solution containing 50 mM potassium phosphate buffer, pH 7.0, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM sodium azide, 1 mM GSH, 1 unit of GSH reductase, and 10-50 µM disecysCD. The mixture was preincubated for 10 min, and then 0.25 mM NADPH solution was added and incubated for 3 min at 37 °C. Thereafter, the reaction was initiated by addition of 0.5 mM hydroperoxides. Organic hydroperoxides were dissolved in methanol, which did not affect the activity assay. The activity was determined by the decrease of NADPH absorption at 340 nm. Appropriate controls were run without enzyme mimic and were subtracted. The activity unit of the mimic is defined as the amount of the mimic that utilizes 1 µmol of NADPH/min. The activity is expressed in units per micromole of enzyme mimic. Assay of Kinetics of DisecysCD. The assay of kinetics of disecysCD was similar to that of native GPX (28). The initial rates were determined by observing the change of NADPH absorption at 340 nm at several concentrations of one substrate while the concentration of the other substrate was kept constant. All kinetic experiments were performed in 0.5 mL of the reaction solution containing 50 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA, 1 unit of GSH reductase, 0.25 mM NADPH, and appropriate concentration of GSH, H2O2, and disecysCD. The reaction conditions and operation
684 Bioconjugate Chem., Vol. 11, No. 5, 2000
Ren et al.
Figure 1. Double-reciprocal plots of the initial velocity vs the concentration of the substrates. (A) [E]0/V0 (min) vs 1/[H2O2] (mM-1) for 2 µM disecysCD, at [GSH] ) 1.0 mM (O), 2.0 mM (0), and 3.0 mM (4). (B) [E]0/V0 (min) vs 1/[GSH] (mM-1) for 10 µM disecysCD, at [H2O2] ) 0.5 mM (O), 1.0 mM (0), and 2.0 mM (4). Table 1. GPX Activities of DisecysCD and Other Species species SeCys Ebselen PhSeSePh DisecysCDa,b β-CDa,b β-CD + SeCysa,b
hydroperoxide H 2 O2 H2O2 H2O2 H2O2 t-BuOOH CuOOH H2O2 H2O2
activity (units/µmol) 0.05 0.99 1.95 4.13 (0.2) 2.11 (0.25) 5.82 (0.25) 0.03 (0.01) 0.11 (0.05)
a
Reactions were carried out in 50 mM potassium phosphate buffer, pH 7.0, at 37 °C, 1 mM GSH, 0.5 mM H2O2. b Standard deviations are shown in parentheses.
procedure were basically same as that described in Determination of the GPX Activity of DisecysCD. The reaction was initiated by addition appropriate concentration of H2O2. The nonenzymatic reaction affecting the measurement of the initial rate was taken into account and subtracted to obtain exact kinetic values. RESULTS
GPX Activities of DisecysCD. The GPX activity was monitored using the modified Wilson’s method. The noenzymatic reaction was taken into account and then subtracted to obtain the exact activity. The GPX activities of the disecysCD-catalyzed reduction of a variety of hydroperoxides by GSH were listed in Table 1 and the GPX activity of other species were included for comparison. From the Table 1, the GPX activity of disecysCD for the reduction of H2O2 by GSH was found to be 4.13 units/ µmol, indicating that disecysCD displays a higher GPX activity than SeCys (5), ebselen (10), and diphenyl diselenide (PhSeSePh) (10). At 1 mM GSH and 0.5 mM H2O2, the initial rates of the reduction of H2O2 by GSH were 9.0 × 10-6 M min-1 for disecysCD (2 µM), 2.1 × 10-6 M min-1 for ebselen (2 µM), 4.7 × 10-7 M min-1 for SeCys (2 µM), 4.2 × 10-7 M min-1 for β-CD (2 µM), and 5.8 × 10-7 M min-1 for β-CD + SeCys (2 µM). The spontaneous reaction rate was determined to be 3.6 × 10-7 M min-1. These data show that disecysCD, ebselen, SeCys, β-CD, and β-CD + SeCys accelerate the reaction by 25-, 6-, 1.3-, 1.2-, and 1.6-fold, respectively. The disecysCD also catalyzes the reduction of organic hydro-
Table 2. Kinetic Parameters for the GPX Activity of DisecysCDa,b [H2O2] (mM)
kcat(app) (min-1)
KGSH(app) (M)
kcat(app)/KGSH(app) (M-1 min-1)
0.5 1.0 2.0
4.8 ( 0.5 5.3 ( 0.2 6.3 ( 0.4
(0.42 ( 0.02) × 10-3 (0.47 ( 0.03) × 10-3 (0.55 ( 0.02) × 10-3
(1.14 ( 0.04) × 104 (1.13 ( 0.08) × 104 (1.15 ( 0.03) × 104
[GSH] (mM)
kcat(app) (min-1)
KH2O2(app) (M)
kcat(app)/KH2O2(app) (M-1 min-1)
1.0 2.0 3.0
2.8 ( 0.2 5.1 ( 0.5 9.1 ( 02
(0.22 ( 0.01) × 10-3 (0.38 ( 0.06) × 10-3 (0.72 ( 0.03) × 10-3
(1.27 ( 0.04) × 104 (1.34 ( 0.01) × 104 (1.26 ( 0.03) × 104
a Reactions were carried out in 50 mM potassium phosphate buffer, pH 7.0, at 37 °C. b The data in the table were obtained from the plots in Figure 1.
peroxides such as t-BuOOH and CuOOH. The spontaneous reaction rates of the reduction of these hydroperoxides were found to be lower than that of H2O2. The GPX activities of disecysCD for reduction of t-BuOOH and CuOOH by GSH were determined to be 2.11 and 5.82 units/µmol, respectively. Kinetics of the DisecysCD-Catalyzed Reduction of H2O2 by GSH. To probe the mechanism of the disecysCD-catalyzed reduction of H2O2 by GSH, comprehensive kinetic studies were carried out. The initial rates for the reduction of H2O2 by GSH were determined as a function of substrate concentration, varying one substrate concentration while the other fixed. Michaelis-Menten kinetics were observed for both substrates under all the conditions investigated. The apparent kinetic parameters obtained at several GSH and H2O2 concentrations are summarized in Table 2. Double-reciprocal plots of the initial velocity vs the concentration of the substrates yielded families of parallel lines for both substrates (Figure 1), consistent with a ping-pong mechanism involving at least one covalent enzyme intermediate. The relevant steady-state equation for this system is
v0 [E]0
)
kmax[GSH][H2O2] KGSH[H2O2] + KH2O2[GSH] + [H2O2][GSH]
(1)
where kmax is a pesudo-first-order rate constant and KH2O2 and KGSH are the Michaelis constants for H2O2 and GSH,
Cyclodextrin Mimic of Glutathione Peroxidase
respectively. From the data in Table 2, kmax ) 14.3 ( 0.8 min-1, KH2O2 ) 2.06 ( 0.15 mM, and KGSH ) 0.59 ( 0.10 mM. Thus, kmax/KH2O2 ) 6.9 × 103 min-1 M-1 and kmax/ KGSH ) 2.4 × 104 min-1 M-1.
Bioconjugate Chem., Vol. 11, No. 5, 2000 685 Scheme 2
DISCUSSION
A coupled test procedure was performed to assay the GPX activities of disecysCD. The oxidized GSH formed in catalytic reaction was reduced to reduced GSH by adding NADPH in the presence of GSH reductase (eqs 2 and 3) enzyme
2GSH + ROOH 98 GSSG + ROH + H2O (2) where R ) H,t-butyl,cumenyl Reductase
GSSG + NADPH + H+ 98 2GSH + NADP+ (3) The amount of oxidized GSH was measured in terms of the decrease of NADPH absorbance at 340 nm. At the NADPH concentration used, the coupled reaction was a zero-order reaction with respect to NADPH and the oxidation of GSH was the rate-limiting step. After adding a variety of hydroperoxides to the reaction solution, a linear-dependent decrease of NADPH was found during the initial 10 min of the reaction. The reaction mixture was preincubated with GSH to obtain the maximum activities. In our experiment, the GPX activities of disecysCD for reduction of H2O2, t-BuOOH, and CuOOH were enhanced 1.5, 1.2, and 1.8-fold by preincubation with GSH, respectively. The only marginal GPX activity was observed in reduction of H2O2 by GSH in the presence of SeCys that is the catalytically active group of the native GPX, and addition of β-CD appreciably enhanced the GPX activity. It should be noted that β-CD itself was as the catalyst, suggesting that the hydrophobic cavity of β-CD play an important in the catalytic cycle. When SeCyss was bound to the primary face of β-CD, the disecysCD-catalyzed reduction of H2O2 by GSH was considerably accelerated and the GPX activity of disecysCD was 82-fold more actives than that of SeCys. Enhancement of rate by disecysCD was explained by correlating the crystal structure of the native enzyme with the properties of β-CD. Clearly, the ability to bind this thiol substrate is essential for the enzyme activity. In the vicinity of the reactive group of the native GPX, some aromatic amino acid residues that can form hydrophobic environment facilitate substrate GSH binding (2). Analysis of the incubation solution containing GSH and cyclodextrin using electrospray mass spectrometry and tandem mass spectrometry reveals that cyclodextrin itself could bind GSH to form intramolecular complex (29). Binding of GSH by 6-OTs-6-deoxy-β-CD was investigated by spectrophotometric titration and binding constant was found to be 1.01 × 102 M. These observations suggest that cyclodextrin and cyclodextrin’s derivative bind GSH to form the intramolecular complex. Cyclodextrins provide hydrophobic cavity to bind the substrate GSH and bring the catalytic group and GSH together, then the initial binding interaction between disecysCD and GSH accelerates the reaction of GSH and H2O2 (19). The hydrophobic action of cavity of β-CD plays a vital role in rate enhancement. So it is not surprising to discover that the GPX activity of conjugate disecysCD was more active than that of SeCys, ebselen, and PhSeSePh. The GPX activity of disecysCD could be enhanced by improving the ability to bind another substrate hydroperoxide. Cyclo-
dextrins have stronger ability to bind shape/size-fit organic molecule (the aromatic group in CuOOH) than that for shape/size-unfit organic molecule (the tert-butyl group in t-BuOOH) in the cavity (30). The substrate CuOOH could take advantage of the cavity of disecysCD more effectively, thus the GPX activity for CuOOH is 2.8fold than that for t-BuOOH. These results show that to bind substrate is a major problem that should be considered in the development of artificial imitation of GPX. The kinetic data for disecysCD can be a triple-transfer mechanism and may be described by Dalziel’s parameter (31).
ΦH2O2 ΦGSH [E]0 ) Φ0 + + v0 [GSH] [H2O2]
(4)
The form of the rate equation obtained is identical with that of native GPX in vivo except for the presence of the Φ0 term, which is negligible in the native GPX cycle. According to the native GPX cycle shown in Scheme 2, one can easily obtain the rate equation described as eq 5 (32).
ΦH2O2 ΦGSH [E]0 ) + ν0 [GSH] [H2O2]
(5)
Similarity of the rate equation between disecysCD and the native GPX clearly suggest that disecysCD may exactly follow the native GPX cycle. An initially slow rate of H2O2 consumption was observed in disecysCD-catalyzed the reduction of H2O2 by GSH without preincubation with GSH. We interpret this rate lag as initially slow reaction of disecysCD and GSH (eq 6).
CDCysSeSeCysCD + GSH T CDCysSeH + CDCysSeSG (6) Indeed, the lag time was reduced as GSH concentration was increased. In the studies of PhSeSePh mechanism, the interconversion of S-(phenylselenyl) glutathione, benzenselenolate, and benzeneselenenic acid has been proposed in the catalytic cycle (33). According to these observations, our disecysCD should adapt the similar mechanism proposed for PhSeSePh. In this mechanism (Scheme 3), the CDCysSeSeCysCD (disecysCD) lies off the main catalytic cycle, and the main catalytic cycle is consistent with the native GPX. The kinetic parameters for the native GPX, acting on H2O2 and GSH, at pH 7.0 and 37 °C, are ΦH2O2 ) 0.93 × 10-10 M min and ΦGSH ) 2.13 × 10-8 M min (28). The equivalent kinetic parameters for disecysCD, acting on H2O2 and GSH, at pH 7.0 and 37 °C, are ΦH2O2 ) 1.5 × 10-4 M min, and ΦGSH ) 4.2 × 10-5 M min, by inspection of eq 1. These parameters can be directly compared, since they were measured at the same pH and temperature for the same substrate. Native GPX is believed to have
686 Bioconjugate Chem., Vol. 11, No. 5, 2000 Scheme 3
evolved to optimal efficiency for the decomposition of H2O2, having an apparent second-order rate constant for the reaction between H2O2 and enzyme of the order of 1010 M-1 min-1. DisecysCD falls far short of this ideal, giving an equivalent rate constant for hydroperoxide consumption of approximately 7 × 103 M-1 min-1. The rate of reaction between the two enzymes and their respective substrate GSH were also different, as evidenced by the values of ΦGSH above. This shows that disecysCD is far less efficient than the native GPX. In the eq 4, Φ0 has a finite value for disecysCD but is equal to zero for GPX. This indicates that there is no accumulation of catalytic intermediates for the native GPX, but such intermediates do build up in case of disecysCD (34). Our kinetic data suggest that the attack of GSH on CDCysSeSG may be rate determining to some degree and hence that CDCysSeSG would be accumulated, which is agreement with the reactivity of the intermediates. Among the intermediates, selenenyl sulfide (CDCysSeSG) could be synthesized and separated (33, 35). Because the concentration of CDCysSeH would be low under steady-state conditions, the low concentration of the selenolate may be responsible for the slow rate of decomposition of H2O2, which reacts with this disecysCD intermediate. On the basis of the discussion above, it may be possible to design an artificial enzyme model, which could make use of the enzyme’s binding energy to conquer the rate-determining step and hence resolve this question. On the basis of above discussions, it is important to consider binding substrate in designing GPX mimics. Our enzyme model, which only has hydrophobic environment, is simpler than native enzyme, which not only has hydrophobic environment (Phe and Trp) but also has two Arg residues and one Gln residue, which form salt-bridge and hydrogen bond with GSH to help binding GSH. On the basis of molecular design, it is possible to obtain strong substrate binding by modification of cyclodextrin overcoming the rate-determining step. The information obtained in this study should be useful for understanding the native enzyme and the design and synthesis of highly active enzyme models with GPX activity. ACKNOWLEDGMENT
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