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Model Study Using Designed Selenopeptides on the Importance of the Catalytic Triad for the Antioxidative Functions of Glutathione Peroxidase Toshiki Takei,† Yoshiko Urabe,† Yuya Asahina,‡ Hironobu Hojo,*,‡,§ Takeshi Nomura,† Kenichi Dedachi,† Kenta Arai,† and Michio Iwaoka*,† †

Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan Department of Applied Biochemistry, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan

‡

S Supporting Information *

ABSTRACT: Although the catalytic triad of glutathione peroxidase (GPx) has been well recognized, there was little evidence for the relevance of the interactions among the triad amino acid residues, i.e., selenocysteine (U), glutamine (Q), and tryptophan (W), to the GPx antioxidative functions. Using a designed selenopeptide having an amino acid sequence of GQAUAWG, we demonstrate here that U, Q, and W present at the active site can interact with each other to exert the enzymatic activity. The amino acid sequence was chosen on the basis of the Monte Carlo molecular simulation for various selenopeptides in polarizable continuous water using the SAAP force field (SAAP-MC). Measurement of the GPx-like activity for the selenopeptide obtained by solidphase peptide synthesis revealed that the antioxidant activity is cooperatively enhanced by the presence of Q and W proximate to U, although the activity was low compared to selenocystine (U2). The effect of Q on the activity was more important than that of W. In addition, the fluorescence spectrometry suggested a close contact between U and W. These experimental observations were supported by SAAP-MC simulation as well as by ab initio calculation. The latter further suggested that the interaction mode among the triad changes depending on the intermediate states.

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INTRODUCTION Glutathione peroxidase (GPx), an antioxidant selenoenzyme widely present in living organisms,1−4 has a selenium (Se) atom at the active site as selenocysteine (Sec; U), which catalyzes the reduction of hydroperoxides, such as H2O2, at the expense of glutathione (GSH), i.e., H2O2 + 2GSH → 2H2O + GSSG. There are three active states of GPx in the catalytic cycle. The selenol state (ESeH), in which the selenol functional group must be deprotonated to selenolate (i.e., ESe−) because of the low pKa value,5 is the standard form under physiological conditions. The reaction of ESeH with H2O2 affords the selenenic acid state (ESeOH), which is rapidly reduced with two molecules of GSH back to ESeH through the selenenyl sulfide state (ESeSG). The X-ray structure analysis of GPx6 as well as the mutational and phylogenetic analyses7−9 revealed the presence of a catalytic triad constituted by Sec (U), Gln (Q), and Trp (W) residues (Figure 1). Although the side-chain N atoms of Q and W were supposed to interact with the Se atom of U in ESeH via two NH···Se hydrogen bonds,8 there was little evidence for the relevance of the interactions to the GPx antioxidative functions. To model the active site structure of GPx, many types of organoselenium compounds have been synthesized to date.10,11 Ebselen,12−14 the typical model, has an isoselenazoline ring, which can be opened by the reaction with thiol to generate the active selenenyl sulfide form.15 Investigation on the antiox© 2013 American Chemical Society

Figure 1. A catalytic triad of GPx. The structure was drawn by using the PDB data (1gp1). Notice that the enzyme is in the stable seleninic acid state (ESeO2H) and that hydrogen atoms are omitted. One of the two independent GPx molecules in the crystal is shown. The interaction parameters for the other molecule are 3.27 and 3.49 Å for U···Q and U···W, respectively, and 71.6° for the N···Se···N angle.

idative catalytic capacity of the amino acid conjugates of ebselen indicated that the individual amino acids introduced to ebselen exhibit different effects on the GPx-like activity.16,17 However, their roles in the catalytic function in terms of the interactions with the Se active center have not been clear. In the meantime, Received: November 20, 2013 Published: December 19, 2013 492

dx.doi.org/10.1021/jp4113975 | J. Phys. Chem. B 2014, 118, 492−500

The Journal of Physical Chemistry B

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freshly prepared 20% piperidine/NMP for 15 min. The resin was then washed with NMP (×5). Fmoc-Trp(Boc)-OBt, which was prepared by mixing Fmoc-Trp(Boc)-OH (120 mg, 200 μmol), 1 M DIC/NMP (0.20 mL), and 1 M HOBt/NMP (0.20 mL) for 30 min, was added to the resin. The mixture was vortexed for 1 h at 50 °C. After the coupling, the resin was washed with MeOH/DCM and NMP (×3). The unreacted amino groups were acetylated by using 10% Ac2O and 5% DIEA in NMP for 5 min. Fmoc-Ala-OH (66 mg, 200 μM) was then introduced to the resin by a similar procedure to yield Fmoc-Ala-Trp(Boc)-Gly-Wang resin. After deprotection of the Fmoc group, the solvent was changed to DCM. FmocSec(MPM)-OBt, which was prepared by mixing FmocSec(MPM)-OH (50 mg, 97 μmol), DIC (33 μL, 200 μmol), and HOBt (29 mg, 200 μmol) in DCM (0.40 mL) for 20 min at room temperature, was added to the resin, and the mixture was vortexed for 30 min at room temperature. After the coupling, the resin was washed with MeOH/DCM and DCM (×3). The unreacted amino groups were acetylated by using 10% Ac2O and 5% DIEA in DCM for 5 min. The resin was washed with DCM (×3) and treated with 20% piperidine/ DCM for 5 min. The deprotection reaction was repeated with the freshly prepared 20% piperidine/DCM for 15 min. The resin was washed with DCM (×5). Fmoc-Ala-OH (83 mg, 260 μmol), Fmoc-Gln(Trt)-OH (160 mg, 260 μmol), and FmocGly-OH (77 mg, 260 μmol) were sequentially introduced to the resin by the DIC-HOBt method in DCM. The Fmoc group of the N-terminal was finally deprotected. The resulting resin was washed with DCM (×3) and ether (×3) and was dried in vacuo to yield H-Gly-Gln(Trt)-Ala-Sec(MPM)-Ala-Trp(Boc)Gly-resin (110 mg). A portion of the obtained resin (9.8 mg) was treated with reagent K (0.30 mL) for 2 h at room temperature. After the removal of TFA by N2 stream, the deprotected peptide was precipitated with ether, washed with ether (×3), and dried in vacuo. To deprotect the MPM group, the crude was further treated with low-acidity TfOH (0.30 mL) at −10 °C for 1 h and then at 4 °C for 2.5 h. The product was precipitated with cold ether and washed with ether (×3). Remaining ether was dried in vacuo. The residue was purified by RP-HPLC to give [H-Gly-Gln-Ala-Sec-Ala-Trp-Gly-OH]2 (1) (560 nmol, 26%). MALDI-TOF-MS (m/z) found: 1477.3, calcd for [M+H]+: 1477.4. AAA Gly4.00Ala3.92Gln2.12. Synthesis of [GQAUAAG]2 (2). A similar procedure was applied for the synthesis of [H-Gly-Gln-Ala-Sec-Ala-Ala-GlyOH]2 (2). Yield 11%. MALDI-TOF-MS (m/z) found: 1247.4, calcd for [M+H]+: 1247.4. AAA Gly4.00Ala6.00Gln2.04. Synthesis of [GAAUAWG]2 (3). A similar procedure was applied for the synthesis of [H-Gly-Ala-Ala-Sec-Ala-Trp-GlyOH]2 (3). Yield 20%. MALDI-TOF-MS (m/z) found: 1362.6, calcd for [M]+: 1362.4. AAA Gly4.00Ala5.60. Synthesis of [GAAUAAG]2 (4). A similar procedure was applied for the synthesis of [H-Gly-Ala-Ala-Sec-Ala-Ala-GlyOH]2 (4). Yield 11%. MALDI-TOF-MS (m/z) found: 1132.7, calcd for [M]+: 1132.3. AAA Gly4.00Ala8.00. Synthesis of GQAAAWG. The standard SPPS protocol using HBTU in DMF was employed. Fmoc-Gly-Wang resin (69 mg, 51 μmol) was weighed into a plastic reaction vessel, where the resin was swelled with NMP for 1 h at room temperature. The resin was treated with 20% piperidine/NMP for 5 min with vortex mixing. The deprotection reaction was repeated with freshly prepared 20% piperidine/NMP for 15 min. The resin was then washed with NMP (×7). Fmoc-

the interactions of Q and W to U were theoretically investigated by using various models. Bayse18 calculated on the simple models, such as the MeSeCl···MeCONH2 molecular complex, at the MP2 level of theory to demonstrate the possible rotation of the side-chain amide group of Q to allow the Se···O interaction in ESeOH. Practical models of the triad structure were examined by Morokuma and Musaev,19,20 who suggested the importance of the backbone N atom of the glycine residue next to U and two water molecules present near the active center, based on the DFT and ONIOM studies. It was also observed that the amide side-chain of Q plays an important role as a proton acceptor in ESeH, while the indole side-chain of W would have a rather small effect probably through the interaction to Q. According to these theoretical investigations, Q must be more important than W for the enzymatic function of GPx. Despite the elaborate study to associate the GPx active site structure with the antioxidative functions, the fundamental role of the catalytic triad has not been well-known due to the difficulty in obtaining reasonable models to probe the structure−function relationship. Luo recently devised engineered selenoabzymes by introducing U, Q, and W into the antibody scFv2F3 at the pertinent positions and showed that the Q and W enhance the GPx activity via functional cooperation with U.21 We are interested in modeling selenoenzyme active sites using selenopeptides. In our previous study on GPx,22 it was found that selenoglutathione (GSeH; γEUG) can be a good GPx mimic, but the primary amino acid sequence around the active site (i.e., SLUGT) did not play important roles in the function. In this context, we have designed herein the selenopeptides that can model the catalytic triad structure of GPx.

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METHODS General. The solid-phase peptide synthesis (SPPS) of selenopeptides was carried out by the Fmoc method using the DIC-HOBt activation. Once selenocysteine (Sec; U) was attached to the peptide, solvent was changed from N-methyl-2pyrrolidone (NMP) to dichloromethane (DCM). Completion of the couplings was assessed by the Kaiser test. The obtained resin was treated with reagent K (TFA:thioanisole:H2O:phenol:3,6-dioxa-1,8-octanedithiol = 82.5:5:5:5:2.5),23 and the mixture was vortexed for 2 h at room temperature. The crude was further treated with low-acidity TfOH (TFA:dimethyl sulfide:m-cresol:TfOH = 5:3:1:1).24 The selenopeptides were characterized by MALDI-TOF-MS, RPHPLC, and amino acid analysis (AAA). The AAA sample was prepared by hydrolyzing the solution with 6 M HCl at 150 °C for 2 h in an evacuated sealed tube. N-(9-Fluorenylmethoxycarbonyl)-Se-(p-methoxyphenylmethyl)-selenocysteine (Fmoc-Sec(MPM)-OH) was synthesized by the literature method.25 The fluorescence spectrum was measured on a Jasco FP-6200 spectrofluorometer by using a quartz cell (light path length = 10 mm) under the conditions of 275 nm for the excitation wavelength, 300−500 nm for the emission wavelength range, 60 nm/min for the scan speed, 1 nm for the bandwidth, 1 s for the response, and 5 nm for the slit widths of both the excitation and emission. Synthesis of [GQAUAWG]2 (1). Fmoc-Gly-Wang resin (68 mg, 51 μmol) was weighed into a plastic reaction vessel, where the resin was swelled with NMP for 1 h at room temperature. The resin was treated with 20% piperidine/NMP for 5 min with vortex mixing. The deprotection reaction was repeated in 493



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Tokai University.27,28 The potential parameters are entirely different from conventional force fields in that a polypeptide is divided to the amino acid units, not to the atomic units, solvent effects are implicitly included in the parameters, and the atomic charges are not constant but variable depending on the conformation of the amino acid units. In SAAPFF, the total potential energy (ETOTAL) for a polypeptide is expressed by

Trp(Boc)-OBTU, which was prepared by mixing FmocTrp(Boc)-OH (110 mg, 200 μmol), 0.5 M HBTU/DMF (380 μL), and DIEA (70 μL) for 3 min at room temperature, was added to the resin. The mixture was vortexed for 12 min at room temperature. After the coupling, the resin was washed with NMP (×3). Fmoc-Ala-OH (70 mg, 210 μmol) was then introduced by the HBTU method in DMF to yield Fmoc-AlaTrp(Boc)-Gly-Wang resin. A portion of the resin (25.5 μmol) was then treated with 20% piperidine in NMP. Fmoc-Ala-OH (40 mg, 120 μmol), Fmoc-Ala-OH (37 mg, 110 μmol), FmocGln(Trt)-OH (65 mg, 100 μmol), and Fmoc-Gly-OH (31 mg, 100 μmol) were sequentially introduced to the resin by the HBTU method in DMF to yield Fmoc-Gly-Gln(Trt)-Ala-AlaAla-Trp(Boc)-Gly-Wang resin. After deprotection of the Nterminal Fmoc group, the resin was washed with DCM (×3) and ether (×3) and was dried in vacuo to yield H-GlyGln(Trt)-Ala-Ala-Ala-Trp(Boc)-Gly resin (58 mg). A portion of the resin (55 mg) was treated with TFA cocktail (TFA:TIS:H2O = 95:2.5:2.5; 1.0 mL) for 2 h at room temperature. After the removal of TFA by N2 stream, the peptide was precipitated with ether, washed with ether (×3), and dried in vacuo. The residue was purified by RP-HPLC to give H-Gly-Gln-Ala-Ala-Ala-Trp-Gly-OH (11.8 μmol, 47%). MALDI-TOF-MS (m/z) found: 660.7, calcd for [M]+: 660.3. AAA Gly2.00Ala3.00Gln1.07. Synthesis of GQACAWG. A similar procedure was applied for the synthesis of H-Gly-Gln-Ala-Cys-Ala-Trp-Gly-OH. Yield 32%. MALDI-TOF-MS (m/z) found: 691.8, calcd for [M+H]+: 692.3. AAA Gly2.00Ala2.06Gln1.08. Synthesis of [GQACAWG]2. A similar procedure was applied for the synthesis of [H-Gly-Gln-Ala-Cys-Ala-Trp-GlyOH]2. The crude H-Gly-Gln-Ala-Cys-Ala-Trp-Gly-OH obtained was dissolved in pH 7.0 phosphate buffer (1.6 mL) containing 6 M guanidine hydrochloride. DMSO (400 μL) was added to the solution. The mixture was reacted for 20 h at 4 °C and then at room temperature for 1 day. Yield 24%. MALDITOF-MS (m/z) found: 1381.0, calcd for [M+H]+: 1380.5. AAA Gly2.00Ala2.11Gln1.12. GPx-Like Activity Measurement of Selenopeptides 1− 4. The GPx-like antioxidant activity of selenopeptides 1−4 was assayed according to the literature.26 A test solution (300 μL), containing 1 mM NADPH, 13.3 mM GSH, and 13.3 U/mL glutathione reductase (GR) in 100 mM phosphate/6 mM EDTA buffer at pH 7.4, was added with the phosphate buffer solution (570 μL) and a 830 μM selenopeptide solution (60 μL) in 50% CH3CN/H2O containing 0.1 M TFA, and the mixture was vortexed. The reaction was initiated by addition of a 3.6 mM H2O2 solution (70 μL) to the mixture solution. The reduction rate of H2O2 was monitored at room temperature by absorption change at 340 nm due to consumption of NADPH, which was added in the assay solution to reduce GSSG (a counterproduct of the H2O2 reduction) to GSH in the presence of GR. Since the solubility of selenopeptides in water was generally low, we employed 50% CH3CN/H2O containing 0.1 M TFA as the solvent. The initial concentrations were [GSH]0 = 4 mM, [H2O2]0 = 0.25 mM, [NADPH]0 = 0.3 mM, [GR] = 4 units/mL, and [selenopeptide] = 50 μM. Selenocystine (Sec2) and PhSeSePh were used as a control of the GPx activity in each set of measurements. The assay was repeated more than five times. Preparation of SAAP Parameters for Sec. The SAAP force field (SAAPFF) is the potential energy function of polypeptide molecules developed in Iwaoka’s laboratory in

ETOTAL = E SAAP + E ES + ELJ + E OTHERS

where ESAAP is a sum of potential energies (SAAP) for the individual amino acid units, EES and ELJ are electrostatic and Lennard-Jones potentials between the amino acid units, and EOTHERS is the other correlation term, which is ignored in a current version of SAAPFF. Detailed descriptions of these terms were given previously.27,28 The SAAPFF has been improved recently.29 The SAAPFF parameters for Sec (U) were developed as follows. On the basis of the framework of the side-chain separation approximation,28,29 the geometry of (CH3)3CCH2Se− was optimized in water (IEFPCM) at the HF/6-31+G(d) level by using the Gaussian 03 program.30 The ESP charges were then obtained at the higher MP2/6-31G(d,p) level in the IEFPCM water. The van der Waals parameters of the Se atom were adopted from the literature.31 Molecular Simulation. SAAP-Monte Carlo (SAAP-MC) simulation was carried out at 300 K in polarizable water. The conventional Metropolis method32,33 and the Mersenne Twister random number generator34 were employed in the MC simulation. At each MC step, one dihedral angle was changed randomly with a maximum displacement angle of ±32°. Five trajectories with 1 billion total MC steps were obtained for each selenopeptide starting from the extended structure. An Intel Xeon W3550 3.06 GHz processor with 12 GB memory was employed as a platform for calculation. The computing time of the SAAP-MC simulation was about 80 h for each trajectory. From each SAAP-MC simulation trajectory, 10 000 structures were extracted in every 100 000 steps. The total 50 000 structures, which were obtained from the five different trajectories, were then statistically analyzed on the basis of the Se···N atomic distances between Sec and Gln side chains or Sec and Trp side chains. On the other hand, the structures obtained from each trajectory were classified to 10 structural clusters by using a clustering algorithm called the kmeans method35 based on the RMSD for all heavy atoms except for the hydrogen atoms. Ab Initio Calculation. The representative structures of GQAUAWG obtained from the structure clustering, which have short atomic contacts for both Se(U)···N(Q) and Se(U)··· W(Q), were further analyzed by ab initio calculation. First, the geometry was fully optimized in vacuo at HF/6-31G(d). Second, to the optimized selenolate structure, a OH or SCH3 group was added to build the selenenic acid or selenenyl sulfide form of GQAUAWG, respectively. Two orientations of the substituent were chosen for each case: one is the backside of the Se(U)···N(Q) contact, and the other is the backside of the Se(U)···W(Q) contact. Then, the obtained structures were processed for full geometry optimization in vacuo at HF/631G(d).

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RESULTS AND DISCUSSION Design of the Amino Acid Sequence of Selenopeptides. To search for the useful amino acid sequence to model the catalytic triad, the Monte Carlo simulation using the SAAP 494



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force field (SAAP-MC) 29 was carried out for several selenopeptides having an amino acid sequence of GQXUXWG, where X is G, A, AA, or AAA. SAAP is the single amino acid potential force field recently developed in our laboratory for molecular simulation of polypeptides using the single amino acid potentials in water.27,28 To perform the simulation, we developed the SAAP force field parameters for U in the selenolate (Se−) form (Figure 2, Table 1). The side-chain

As for the Se···N distance between U and Q (Figure 4a), the short contacts (∼4 Å) were most frequently observed for GQAUAWG, while the distance became significantly longer when the spacer amino acid (X) was G, suggesting that spacer A possesses adequate flexibility to maintain the close contact between U and Q. Since the vdw radii of Se and N are 1.9 and 1.55 Å, respectively,36 there should not be direct Se···N contacts but hydrogen-bonding interactions between the two close atoms. On the other hand, the ratio of the short Se···N contacts gradually decreased with increasing number of spacer A. Similar trends were observed for the Se···N distance between U and W (Figure 4b), although the short Se···N contacts were most frequently observed with the spacer of AA. The results suggested that GQAUAAWG may be a good model to mimic the triad. However, the SAAP-MC simulation for this sequence showed a decrease in the ratio of the structures with short atomic contacts for the Se···N pairs. Considering the presence of a small maximum peak at 4 Å in the distribution of the Se··· N distance between U and W (Figure 4b) as well as easiness in the synthesis of short selenopeptides, we chose GQAUAWG as a promising model. It should be noted that GWAUAQG with the reverse amino acid sequence did not show close Se···N contacts. Assessment of GPx-Like Antioxidant Capacity. According to the SAAP-MC simulation results, we synthesized selenopeptide models 1−4, having a diselenide (SeSe) linkage, by solid-phase peptide synthesis (SPPS) applying the Fmoc strategy.37 The identity and purity of the dimer selenopeptides were unambiguously characterized by amino acid analysis as well as MS and HPLC analyses. The GPx-like antioxidant activity of these precatalysts was then evaluated by using the NADPH-coupled assay in the presence of glutathione reductase (GR).26 In this assay, the initial velocities of the consumption of NADPH were monitored by the UV absorption change at 340 nm in the presence of GR, GSH, H2O2, and the SeSe precatalyst, which can be activated by the reaction with H2O2 or GSH to participate in the catalytic cycle.38 GSSG produced by the reaction of H2O2 and GSH is reduced with NADPH back to GSH by the function of GR. Thus, the decrease of NADPH

Figure 2. Molecular structure of the Sec (U) side-chain for SAAPFF parameters.

structure of U and the electrostatic potential (ESP) atomic charges were obtained in water by ab initio calculation applying the polarizable continuum model (PCM) according to the literature.29 The van der Waals parameters for the Se atom were adopted from the literature values determined by Prabhakar et al.31 SAAP-MC simulation for GQXUXWG was performed with 1 billion MC steps at 300 K in PCM water. Five trajectories were obtained starting from the same extended structure using different random number seeds. One of the energetic trajectories obtained for GQAUAWG is shown in Figure 3 along with the changes of Se···N distances and the all-atom RMSD (expect for H), which was calculated based on the structure obtained by ab initio calculation (vide infra). The distributions of the intramolecular Se···N atomic distance between U and Q or U and W for the obtained 50 000 structures are shown in Figure 4. Table 1. SAAPFF Parameters for Sec (U)

a

no.

atom

x

y

z

qa

Rb

εc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

C Se H H C C C C H H H H H H H H H

0.0000 0.9794 0.3437 0.3437 −1.5698 −2.0658 −2.2000 −2.0658 −1.7243 −1.6857 −3.1541 −1.8838 −1.8838 −3.2893 −1.6857 −1.7243 −3.1541

0.0000 1.7281 −0.5379 −0.5379 0.0000 −0.7633 1.3965 −0.7633 −0.2744 −1.7827 −0.8136 1.9624 1.9624 1.3167 −1.7827 −0.2744 −0.8136

0.0000 0.0000 −0.8753 0.8753 0.0000 −1.2420 −0.0000 1.2420 −2.1507 −1.2532 −1.2777 0.8662 −0.8662 −0.0000 1.2532 2.1507 1.2777

−0.5479 −1.0332 0.1628 0.1628 0.8850 −0.4720 −0.3898 −0.4721 0.0949 0.0756 0.0824 0.0781 0.0780 0.0426 0.0756 0.0950 0.0824

1.9080 2.1000 1.3870 1.3870

0.1094 0.2910 0.0157 0.0157

Atomic charges (electron). bAtomic radius (Å). cThe depth of Lennard−Jones potential well (kcal/mol). 495



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Figure 3. One of the five SAAP-MC simulation trajectories for GQAUAWG. (a) The potential energy trajectory. (b) The trajectory of the Se(U)··· N(Q) distance. (c) The trajectory of the Se(U)···N(W) distance. (d) The trajectory of the RMSD value for all atoms except for H from the structure (shown in Figure 6) obtained by ab initio calculation with full geometry optimization.

Table 2. Initial Velocities (v0) of the H2O2 Reduction Observed in the NADPH-Coupled GPx Assay Se catalysts

v0a (μM/min)

blank Sec2 [GQAUAWG]2 (1) [GQAUAAG]2 (2) [GAAUAWG]2 (3) [GAAUAAG]2 (4)

± ± ± ± ± ±

10 112 37 33 19 23

relative activityb

c

2 10c 3 5 1 2

1 0.33 0.30 0.17 0.20

(1.6) (1.4) (0.8) (1.0)

a

Reaction conditions: [GSH]0 = 4 mM, [H2O2]0 = 0.25 mM, [NADPH]0 = 0.3 mM, [GR] = 4 units/mL, and [Se catalyst] = 50 μM in pH 7.4 phosphate buffer at room temperature. bThe relative GPxlike activity with respect to Sec2. The values in parentheses are the relative activity to selenopeptide 4. cData from ref 22. Figure 4. Distributions of the Se···N distances in 50 000 structures obtained by SAAP-MC simulation in water at 300 K: (a) the Se(U)··· N(Q) distance; (b) the Se(U)···N(W) distance.

1.4), whereas 3 with W had lower activity than 4 (relative activity 0.8), suggesting that Q enhances the GPx activity while W decreases it. On the other hand, 1 with both Q and W showed the highest antioxidant activity among the four selenopeptides (relative activity 1.6). The result clearly demonstrated the cooperative effect of the Q and W residues on the activity, supporting the importance of the active site triad on the GPx activity. The apparently smaller contribution of W than Q to the activity is consistent with the results from the previous DFT and ONIOM calculations.19,20 Furthermore, the trends of the activity observed for selenopeptides 1−4 are similar to those recently reported for engineered selenoabzymes with site-directed muatations:21 The relative GPx activity for the selenoabzyme with mutations of U, Q, and W was ∼1.8 with respect to the one with a mutation of only U. The similarity between the selenoabzymes and our short selenopep-

corresponds to the reduction rate of H2O2. The observed initial velocities (v0) of H2O2 reduction are listed in Table 2. Although the catalytic activities of selenopeptides 1−4 were lower than that of selenocystine (Sec2), which is an oxidized dimer of U, all the selenopeptides showed GPx-like antioxidant activity with respect to the blank. In our previous study,22 there was a trend that the GPx-like activity decreases with increasing chain length of selenopeptides: the v0 values were 44 and 39 μM/min for [LUG]2 and [SLUGT]2, respectively. In light of this trend, the GPx-like activities observed for heptapeptides 1− 4 are in a reasonable range. However, it is notable that the activities were sequence-dependent. The activity of 2 with Q was higher than that of 4 without Q and W (relative activity 496



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tide models may indicate that the capacity of the GPx catalytic triad to control the reactivity of the Se reaction center does not change significantly by the type or size of the scaffold around the triad. Conformational Insight from Fluorescence Spectroscopy. The evidence for the close contact between U and W was obtained by fluorescence spectrometry (Figure 5). Since W

Figure 6. The structure of GQAUAWG obtained by SAAP-MC simulation and subsequent ab initio calculation with geometry optimization at HF/6-31G(d). Most of the hydrogen atoms are omitted for clarity except for the Q2 amide and W6 indole groups. The atomic distances for Se(U4)···N(Q2) and Se(U4)···N(W6) were 3.62 and 3.52 Å, respectively. The N(Q2)···Se(U4)···N(W6) angle was 74.5°.

Ab initio calculation was further carried out for selenenic acid GQAU(SeOH)AWG and selenenyl sulfide GQAU(SeSMe)AWG by modifying the Se atom of GQAUAWG (Figure 6) with a OH or SMe group, respectively. These groups were attached to the Se atom in two different orientations, i.e., in the backside of Se(U)···N(Q) (structure A) or Se(U)···N(W) (structure B). Figure 7 shows the structures of the selenenic

Figure 5. Fluorescence spectra of (a) 1 (20 μM), (b) GQACAWG (C = cystine) (20 μM), (c) GQACAWG (C = cysteine) (40 μM), and (d) GQAAAWG (27 μM) in pH 7.4 phosphate buffer at room temperature.

has an indole ring, 1 showed a strong emission peak at 360 nm when it was flashed with the UV at 275 nm. However, it appeared that the fluorescence intensity is significantly suppressed compared with those of GQAAAWG and GQACAWG (C = cysteine or cystine) without U. Since the fluorescence intensity of an indole ring is usually decreased by the interaction with neighbor functional groups,39 the result strongly suggested the presence of the interaction between U and W for 1 in the SeSe form. Interaction Modes among the Triad. A protocol of SAAP-MC simulation with structure clustering and subsequent ab initio calculation has recently been shown to be an effective strategy toward structure prediction for short peptide molecules, although the relative energy obtained by SAAPMC is not yet satisfactory.29 Therefore, structural clustering was performed for the structures that were obtained for GQAUAWG by SAAP-MC simulation. As a result, the three representative structures having short atomic contacts for both Se(U)···N(Q) and Se(U)···N(W) were found in 1.4% among 50 000 structures. It should be noted that the low population does not necessarily reflect the relative energy because several approximations have been adopted by the current version of the SAAP force field.29 The obtained structures were then fully optimized by ab initio calculation. The most stable structure is shown in Figure 6. It is notable that the selenolate (Se−) retained the interactions through formation of the two NH···Se hydrogen bonds, suggesting that at least some portion of GQAUAWG would have similar conformations in solution. In the optimized structure of GQAUAWG (Figure 6), which corresponds to the selenolate form of 1, the Se···N distances and the N···Se···N angle are similar to the X-ray structure (Figure 1) although the Se atom in the X-ray structure is in the oxidized seleninic acid form (ESeO2H).6 The observed structural similarity supports the adequacy of 1 as a reasonable GPx triad model as well as the assumption that the interaction modes in the ESeH state are NH···Se hydrogen bonds between U···Q and U···W.

Figure 7. Optimized structures of GQAU(SeOH)AWG at HF/631G(d). Most of the hydrogen atoms are omitted for clarity except for the Q2 amide and W6 indole groups. Structure A: The atomic distances for Se(U4)···O(Q2), Se(U4)···N(W6), and Se(U4)···C(W6) were 3.30, 3.97, and 3.88 Å, respectively. The N(W6)···O(Q2) distance was 3.15 Å. Structure B: The atomic distances for Se(U4)··· C(W6) and Se(U4)···O(A5) were 4.22 and 3.12 Å, respectively. The N(W6)···O(Q2) distance was 2.97 Å.

acid obtained after geometry optimization at HF/6-31G(d). It is seen that the indole ring of W tends to remain in the proximity of Se(U), although the interaction mode is switched from NH···Se to CH···Se hydrogen bond. On the other hand, the amide group of Q is rotated and pushed a little away from U. In structure A, a direct atomic interaction is found between the amide O(Q2) and Se(U4) atoms.40 Such conformational changes were in reasonable agreement with Bayse’s calculation.18 In structure B, on the other hand, Se···O is formed between the main chain O(A5) and Se(U4) atoms. The interaction networks around the Se atom would control the undesirable decomposition or further oxidation by stabilizing the highly reactive SeOH moiety. The structure obtained for GQAU(SeSMe)AWG by ab initio calculation is shown in Figure 8. Although the two different orientations of the SMe moiety were applied as the initial structures, they converged to the same structure after geometry optimization. It is seen that the close contact between the Se 497



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revealed that the conformation having the short Se···N distances between both U···Q and U···W residues would exist in the SeH state. The mode of the interactions in ESeH must be the formation of NH···Se hydrogen bonds to the selenolate center.8 In ESeOH and ESeSG, on the other hand, the interaction mode would become different: the indole ring of W may keep a close contact to U through a CH···Se hydrogen bond, while the amide group of Q would be rotated and pushed a little away from U, presumably stabilizing the resulting intermediate states by formation of weak Se···O interaction instead of NH···Se hydrogen bond.18 The close contact of the indole···Se interaction during the catalytic cycle was reasonably supported by the fluorescence spectra (Figure 5). Selenopeptides are attracting increasing interest in their applications to protein folding 41−43 as well as structural analysis of proteins.44,45 Our results suggest that, in addition to these applications, short selenopeptides are useful tools to model the function of selenoenzymes.

Figure 8. An optimized structure of GQAU(SeSMe)AWG at HF/631G(d). Most of the hydrogen atoms are omitted for clarity except for the Q2 amide and W6 indole groups The atomic distances for Se(U4)···O(Q2), Se(U4)···C(W6), and Se(U4)···O(U4) were 3.33, 3.81, and 3.12 Å, respectively. The N(W6)···O(Q2) distance was 3.08 Å.

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ASSOCIATED CONTENT

S Supporting Information *

Characterization and GPx assay of selenopeptides 1−4, detailed results of SAAP-MC simulation and ab initio calculation, and a complete author list of ref 30. This material is available free of charge via the Internet at http://pubs.acs.org.

atom and the indole group remains through the formation of a CH···Se hydrogen bond as observed for GQAU(SeOH)AWG (Figure 7). Two additional Se···O interactions are present, which are formed in the backsides of Se−S and Se−C covalent bonds as usually observed in such non-bonded interactions.40 It should be noted that the amide group of Q is again rotated to form direct Se···O interaction. The conformational changes observed for GQAU(SeOH)AWG and GQAU(SeSMe)AWG in the ab initio calculation can be rationalized by a loss of the negative charge on the Se atom, which would release the polar amide group of Q more easily than the aromatic indole ring of W. This conformational change may allow the rotation of the amide group of Q so as to stabilize the resulting intermediate states by formation of Se···O interaction. The persistence of the close indole···Se interaction in GQAU(SeSMe)AWG is consistent with the significant quenching of the fluorescence of the indole ring observed for 1, which has an analogous SeSe bond instead of the SeS bond. The interaction networks would consolidate the position of the Se atom at the active site so that the nucleophilic approach of the second GSH molecule to the SeSG state occurs smoothly on the S atom.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H.H.). *E-mail: [email protected] (M.I.). Present Address

§ Institute for Protein Research, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.A. thanks Japanese Society for the Promotion of Science for the Research Fellowship for Young Scientists. The authors thank T. Shimosato for his assistance in developing and maintaining SAAPFF parameters. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research (C): No. 23550198).

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CONCLUSIONS Modeling the amino acid sequence of selenopeptides by applying SAAP-MC simulation and evaluating the GPx-like antioxidative function of the synthesized selenopeptides, we demonstrated here that U, Q, and W can interact with each other to exert the GPx-like antioxidant activity. The effect of Q would be more important than that of W, but the capacity of the selenopeptides as GPx mimics is cooperatively enhanced by the presence of proximate W. The result strongly supported the importance of the catalytic triad in the antioxidative function of GPx. The trend and the range of the relative GPx activity were in reasonable agreement with the recent results obtained for engineered selenoabzymes.21 The modes of the U···Q and U···W interactions are not yet definitive, but the theoretical calculation has provided some useful information. Clustering of the structures obtained by SAAP-MC simulation for GQAUAWG and subsequent geometry optimization of the structures by ab initio calculation

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