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Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond
Biochimica et Biophysica Acta 1547 (2001) 214^220 www.bba-direct.com
Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disul¢de bond Tsutomu Kabashima, Yi Li, Naota Kanada, Kiyoshi Ito, Tadashi Yoshimoto * School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Received 23 January 2001; received in revised form 8 March 2001; accepted 14 March 2001
Abstract From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability. Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis. The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS^PAGE with and without 2-mercaptoethanol. The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. However, the thermal stability of the S185C mutant was found to be 30³C higher than that of wild-type. Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Pyroglutamyl peptidase; Thermal stability; Disul¢de bond; Subunit; Mutagenesis
1. Introduction Pyroglutamyl peptidase (PGP; EC 3.4.19.3) removes amino-terminal pyroglutamic acids (pGlu) from peptides and proteins. The enzymatic activity was previously reported to be present in several bacteria, plants, and animal tissues [1^5]. PGPs can be subdivided into two classes on a mechanistic basis. The type I class consists of mammalian and bacterial PGP that are all cysteine protease type enzymes. The type I enzyme degrades a broad spectrum of pGlu-
Abbreviations: PGP, pyroglutamyl peptidase; pGlu, pyroglutamic acid; 2NNap, L-naphthylamine; 2-ME, 2-mercaptoethanol * Corresponding author. Fax: +81-95-843-2444; E-mail: [email protected]
containing peptides including thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone (LH-RH), bombesin, neurotensin and gastrins [6,7]. The type II enzyme is an incompletely characterized mammalian protein that appears to be a membrane-bound metalloprotease localized in various tissues including muscle, cerebral cortex, and brain [6,8,9]. In contrast to the broad speci¢city of type I enzyme, type II enzyme is highly speci¢c for TRH. Both classes of the mammalian enzymes have been implicated in the regulation of neuropeptide activity, for example in the TRH pathway [10], and in the neurotensin system [11]. Although the role of bacterial enzymes still remains unclear, it has been proposed that PGP activity serves to reduce the toxicity of N-terminally blocked peptides [6], or plays a role in nutrient assimilation [1].
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Fig. 1. Structure of pyroglutamyl peptidases I from B. amyloliquefaciens (1) and T. litoralis (2). Secondary structural elements are shown in a di¡erent color for each subunit. The subunits are named A to D. (1) BPGP: Cys68, Cys144 and Ser185, and (2) TPGP: Cys143 and Cys190 are drawn as ball-and-stick.
We reported the cloning of the PGP gene from Bacillus amyloliquefaciens (BPGP), characterization of its enzymatic properties [12], the three-dimensional structure of the enzyme (Fig. 1 (1)) [13], and the mechanism of substrate recognition [14].
Recently, Singleton et al. reported the crystal structure of the PGP from the hyperthermophilic archaeon Thermococcus litoralis (TPGP) [15]. TPGP showed enhanced thermal stability when compared with enzymes from mesophilic bacteria. TPGP is
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also a homotetramer, and its three-dimensional structure is similar to that of BPGP (Fig. 1 (2)). However, there were two disul¢de bonds between the subunits (A-ss-B, C-ss-D) in TPGP, but not in BPGP. This intersubunit disul¢de bond seems to be responsible for the thermal stability of the TPGP. We found that the introduction of the intersubunit disul¢de bond enhanced the thermal stability of BPGP without any change in its catalytic e¤ciency. 2. Materials and methods 2.1. Materials Restriction endonucleases and various DNA-modifying enzymes were obtained from New England Biolabs or Toyobo Biochemicals. Pyroglutamyl-Lnaphthylamide (pGlu-2NNap) and Fast Garnet GBC were from Sigma. The oligonucleotide primer for site-directed mutagenesis was synthesized by Amersham Pharmacia Biotech. The Mutan-Super Express Km kit and plasmid pKF18k were from Takara Shuzo. The ALF express AutoCycle sequencing kit and other reagents for DNA sequencing were obtained from Amersham Pharmacia Biotech. 2.2. Bacterial strains, plasmids, and medium Escherichia coli MV1184 was used for site-directed mutagenesis. E. coli DH1, JM83, and XLI-Blue were used as a host for expression. Plasmids (pUC18 and pKF18k) were used for expression and mutagenesis as a vector. Bacteria were grown in Luria^Bertani broth (LB broth). 2.3. Site-directed mutagenesis Site-directed mutagenesis was performed according to ODA (Oligonucleotide-directed Dual Amber)-LA PCR method using a Mutan-Super Express Km kit [16,17]. Oligonucleotide primer 5P-CCGAGCCTCTGCCTTGATCAC-3P was used to replace Ser185 with Cys. PCR was carried out in a 50 Wl reaction mixture containing 5 pmol selection primer, 5 pmol mutation
primer, 10 ng of template DNA, 5 Wl of 10ULA PCR Bu¡er II (Mg2 plus), 8 Wl of deoxynucleoside triphosphate (dNTP) mixture at 2.5 mM each, and 2.5 units of TaKaRa LA Taq DNA polymerase. Ampli¢cation involved 30 cycles of denaturation at 94³C for 1 min, annealing at 55³C for 1 min and extension at 72³C for 4 min. After PCR, the reaction mixture was allowed to cool at 4³C. The DNA in the reaction mixture was precipitated by adding 5 Wl of 3 M sodium acetate and 125 Wl of cold 100% ethanol. The precipitate was washed twice with cold 70% ethanol, dried and dissolved in 5 Wl of sterilized water. This DNA solution containing plasmid DNA was used to transform E. coli MV1184, and the transformants were selected on LB plates containing 50 Wg/ml kanamycin. To obtain the C68S/S185C mutant, we used the NruI site between C68 and S185, which is absent from pUC18 vector. The C68S fragment was obtained by digesting pBPG5Cys68Ser [12] with EcoRI and NruI. It was extracted from the agarose gel using the QIAEX II agarose gel extraction kit (Qiagen), and substituted for the same fragment from pBPG5Ser185Cys to produce C68S/S185C double mutant. Mutation was con¢rmed by sequence analysis with the dideoxy chain termination method. Double-stranded plasmids as templates and pUC18speci¢c primers were used. The dideoxy sequencing reactions were performed using an AutoCycle sequencing kit. DNA fragments containing the mutation were prepared by digesting the respective plasmids with EcoRI and HindIII. These fragments were inserted into the same site of pUC18, and then cloned plasmids were used to transform E. coli DH1 or E. coli JM83 for protein expression. 2.4. Puri¢cation of wild-type and mutant enzymes Wild-type and mutant BPGP were puri¢ed as previously described [12]. E. coli DH1 or E. coli JM83 cells harboring a mutant pgp gene plasmid were aerobically cultured in 250 ml of LB medium containing ampicillin at 37³C for 17 h. Cells were harvested by centrifugation (8000Ug; 20 min), washed and resuspended in 20 mM Tris^HCl bu¡er (pH 7.0). The cells were sonicated (ultrasonic disruptor UD200, Tomy) in 30 ml of the above bu¡er. The cell
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Fig. 2. Alignment of pyroglutamyl peptidase sequences. Homology was 34% between the enzymes. B.a, B. amyloliquefaciens; T.l, T. litoralis.
debris was eliminated by centrifugation. The supernatant was fractionated with ammonium sulfate from 40% to 80% saturation, and resuspended in 20 mM Tris^HCl bu¡er (pH 7.0) containing 40% saturated ammonium sulfate. The solution was applied to a hydrophobic column of Toyopearl HW65C equilibrated with 20 mM Tris^HCl bu¡er (pH 7.0) containing 40% saturated ammonium sulfate. The column was washed with the above bu¡er, and the absorbed enzyme was eluted with decreasing linear gradient of ammonium sulfate, from 40% to 0% saturation, in 20 mM Tris^HCl bu¡er (pH 7.0). The active fractions were combined and concentrated by ultra¢ltration using an Amicon apparatus (PM-10) and desalted by dialyzing against 20 mM Tris^HCl bu¡er (pH 7.0) and Milli-Q water. The introduction of a disul¢de bond into the mutant enzyme was con¢rmed by 10% SDS^PAGE under reducing and oxidizing conditions.
scribed previously [12]. To 0.8 ml of 20 mM Tris^ HCl bu¡er (pH 7.0) were added 0.1 ml of 5.0 mM pGlu-2NNap dissolved in 10% dimethyl formamide and 0.1 ml of enzyme solution. After 5 min incubation at 37³C, the enzyme reaction was stopped by adding 0.5 ml of a Fast Garnet GBC (1 mg/ml) solution containing 10% Triton X-100 in 1 M acetate bu¡er (pH 4.0). The absorbance at 550 nm was measured after 15 min, using a Hitachi Model 100-20 or U-3000 spectrophotometer. One unit of activity was de¢ned as the amount of enzyme which released 1 Wmol of 2NNap per min under the above conditions. To determine the Km values, several concentrations of substrate, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 and 3.0 mM, were used. Lineweaver^Burk plots were made to calculate Km and apparent Vmax . To calculate kcat , the subunit molecular weight of wild-type BPGP, 23 286 was used.
2.5. Assay of enzyme activity
2.6. Thermal stability of the mutant enzymes
Pyroglutamyl peptidase activity was assayed spectrophotometrically by the release of L-naphthylamine due to enzymatic hydrolysis of pGlu-2NNap, as de-
The mutant enzymes and wild-type enzyme were preincubated at various temperatures for 30 min, and the remaining activity was measured at 37³C.
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3. Results and discussions 3.1. Intersubunit disul¢de bond formation From the comparison of the three-dimensional structure between BPGP [13] and TPGP [15], it was assumed that the higher thermal stability of TPGP arises from the presence of intersubunit disul¢de bridges. These two enzymes are homotetramers, and their three-dimensional structures are similar. However, an intersubunit disul¢de bond is formed from the Cys190 in TPGP, while it did not exist in BPGP. Amino acid sequence alignment around the Cys190 of TPGP is shown in Fig. 2. Since the Ser185 residue is corresponded in BPGP, we replaced the Ser185 with a Cys residue. The BPGP has two Cys residues (Cys68 and Cys144) per subunit molecule [12]. Cys144 formed the catalytic triad with Glu81 and His168, and the substitution of Cys144 with Ser resulted in a complete loss of activity [12,13]. However, the substitu-
Fig. 4. Thermal stability of the wild-type and mutant BPGPs. The enzyme (8.7 WM) in 20 mM Tris^HCl bu¡er (pH 7.0) was incubated at the indicated temperatures for 30 min, and the residual activities were assayed. a, wild-type; b, S185C; O, C68S/S185C.
tion of Cys68 with Ser did not a¡ect the activity at all [12]. To avoid the formation of the undesirable disul¢de bond between Cys68 and the replaced Cys185, in addition to the S185C mutant, the C68S/S185C double mutant was also constructed. Two mutant enzymes were constructed and could successfully be expressed in E. coli. Wild-type, S185C, and C68S/S185C mutant enzymes were similar with respect to expression levels and yields of puri¢cation (1.5 mg from 250 ml culture). Two mutant enzymes were analyzed by SDS^PAGE with and without 2-mercaptoethanol (2-ME). As shown in Fig. 3, the molecular mass of the wild-type enzyme was about 30 kDa with and without 2-ME. However, the molecular masses of both mutants were about 30 and 60 kDa, with and without 2-ME, respectively. These results suggested that the disul¢de bridge beTable 1 Kinetic constants of wild-type and mutant BPGPs Fig. 3. Con¢rmation of an intersubunit disul¢de bond by SDS^ PAGE. Lanes: 1 and 4, wild-type enzymes; 2 and 5, S185C mutant enzymes; 3 and 6, C68S/S185C mutant enzymes. Lanes 1^3 contained 1% 2-ME in the SDS loading bu¡er as a reducing agent, but in lanes 4^6 it was omitted.
Wild-type S185C C68S/S185C
Km (mM)
kcat (s31 )
kcat /Km (s31 mM31 )
0.25 0.18 0.16
785 770 641
3140 4278 4006
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Fig. 5. E¡ects of pH on the stability. The enzyme was preincubated for 30 min at 30³C and the indicated pH values, and the activity remaining was assayed. a, wild-type; b, S185C.
tween the substituted Cys185 bonded two subunits. Without 2-ME, a faster migrating band close to the 60 kDa band appeared. There is a possibility that cysteine at the active site also formed a disul¢de bridge in a dimer. 3.2. Kinetic parameters of mutant enzymes The kinetic constants, kcat and Km , of the enzymes were measured at 37³C using pGlu-2NNap as substrate. As shown in Table 1, the kcat /Km of the two mutant enzymes for pGlu-2NNap was similar to that of the wild-type. This result indicated that the introduction of the disul¢de bond did not a¡ect the catalytic e¤ciency of the enzyme. 3.3. Physicochemical properties The thermal stability of the wild-type and mutant enzymes was investigated in terms of the activity remaining after incubation at various temperatures. The mutant enzymes showed enhanced thermal stability compared to the wild-type (Fig. 4). Fifty percent remaining activity of the two mutant enzymes was attained at a temperature approx. 30³C higher than that of wild-type enzyme. However, the
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wild-type and mutant enzymes showed no di¡erence in thermal stability under reducing conditions with 1 mM 1,4-dithio-DL-threitol. Therefore, the enhanced thermal stability was due to the introduction of the intersubunit disul¢de bond. The stability of the mutant enzymes compared to the wild-type under various pH conditions was also investigated. Both types showed the same stability in the range 6^8, and retained more than 80% of maximal activity. The S185C mutant enzyme was more stable than the wild-type enzyme at pH 4 or 12 (Fig. 5). Disul¢de bonds can make considerable contributions to stability, an e¡ect mainly attributed to the decreased entropy of the denatured protein [18^20]. Many attempts have been made to increase protein stability by introducing `intramolecular' disul¢de bonds [19,21^24]. In the present study we succeeded to stabilize the BPGP by the introduction of `intermolecular' disul¢de bonds. This seems to be the ¢rst report that intersubunit disul¢de bonds can impart thermal stability in oligomeric enzymes. References [1] R.F. Doolittle, R.W. Armentrout, Biochemistry 7 (1968) 516^521. [2] A. Szewczuk, M. Mulczyk, Eur. J. Biochem. 8 (1969) 63^67. [3] D. Tsuru, K. Fujiwara, K. Kado, J. Biochem. 84 (1978) 467^ 476. [4] W.L. Taylor, J.E. Dixon, J. Biol. Chem. 253 (1978) 6934^ 6940. [5] D. Tsuru, K. Sakabe, T. Yoshimoto, K. Fujiwara, J. Pharmacobiodyn. 5 (1982) 859^868. [6] A.C. Awade, P. Cleuziat, T. Gonzales, J. Robert-Baudouy, Proteins 20 (1994) 34^51. [7] K. Fujiwara, R. Kobayashi, D. Tsuru, Biochim. Biophys. Acta 570 (1979) 140^148. [8] G. Czekay, K. Bauer, Biochem. J. 290 (1993) 921^926. [9] S. Wilk, E.K. Wilk, Neurochem. Int. 15 (1989) 81^90. [10] J. Boler, F. Enzmann, K. Folkers, C.Y. Bowers, A.V. Schally, Biochem. Biophys. Res. Commun. 37 (1969) 705^ 710. [11] T. Nakajima, T. Taminura, J.J. Pisano, Fed. Proc. 29 (1970) 282. [12] T. Yoshimoto, T. Shimoda, A. Kitazono, T. Kabashima, K. Ito, D. Tsuru, J. Biochem. 113 (1993) 67^73. [13] Y. Odagaki, A. Hayashi, K. Okada, K. Hirotsu, T. Kabashima, K. Ito, T. Yoshimoto, D. Tsuru, M. Sato, J. Clardy, Structure 7 (1999) 399^411. [14] K. Ito, T. Inoue, T. Takahashi, H-S. Huang, T. Esumi, S.
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[15] [16] [17] [18] [19] [20]
T. Kabashima et al. / Biochimica et Biophysica Acta 1547 (2001) 214^220 Hatakeyama, N. Tanaka, K.T. Nakamura, T. Yoshimoto, J. Biol. Chem. (2001) in press. M. Singleton, M. Isupov, J. Littlechild, Structure 7 (1999) 237^244. T. Hashimoto-Gotoh, T. Mizuno, Y. Ogasahara, M. Nakagawa, Gene 152 (1995) 271^275. T.A. Kunkel, Proc. Natl. Acad. Sci. USA 82 (1985) 488^492. J.M. Thornton, J. Mol. Biol. 151 (1988) 261^287. C.N. Pace, G.R. Grimsley, J.A. Thomson, B.J. Barnett, J. Biol. Chem. 263 (1988) 11820^11825. T. Zhang, E. Bertelsen, T. Alber, Nat. Struct. Biol. 1 (1994) 434^438.
[21] H. Takagi, T. Takahashi, H. Momose, M. Inouye, Y. Maeda, H. Matsuzawa, T. Ohta, J. Biol. Chem. 265 (1990) 6874^ 6878. [22] J.H. Ko, W.H. Jang, E.K. Kim, H.B. Lee, K.D. Park, J.H. Chung, O.J. Yoo, Biochem. Biophys. Res. Commun. 221 (1996) 631^635. [23] J. Mansfeld, G. Vriend, B.W. Dijkstra, O.R. Veltman, B. Van den Burg, G. Venema, R. Ulbrich-Hofmann, V.G. Eijsink, J. Biol. Chem. 272 (1997) 11152^11156. [24] H. Takagi, K. Hirai, M. Wada, S. Nakamori, J. Biochem. 128 (2000) 585^589.
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Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disul¢de bond Tsutomu Kabashima, Yi Li, Naota Kanada, Kiyoshi Ito, Tadashi Yoshimoto * School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Received 23 January 2001; received in revised form 8 March 2001; accepted 14 March 2001
Abstract From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability. Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis. The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS^PAGE with and without 2-mercaptoethanol. The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. However, the thermal stability of the S185C mutant was found to be 30³C higher than that of wild-type. Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Pyroglutamyl peptidase; Thermal stability; Disul¢de bond; Subunit; Mutagenesis
1. Introduction Pyroglutamyl peptidase (PGP; EC 3.4.19.3) removes amino-terminal pyroglutamic acids (pGlu) from peptides and proteins. The enzymatic activity was previously reported to be present in several bacteria, plants, and animal tissues [1^5]. PGPs can be subdivided into two classes on a mechanistic basis. The type I class consists of mammalian and bacterial PGP that are all cysteine protease type enzymes. The type I enzyme degrades a broad spectrum of pGlu-
Abbreviations: PGP, pyroglutamyl peptidase; pGlu, pyroglutamic acid; 2NNap, L-naphthylamine; 2-ME, 2-mercaptoethanol * Corresponding author. Fax: +81-95-843-2444; E-mail: [email protected]
containing peptides including thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone (LH-RH), bombesin, neurotensin and gastrins [6,7]. The type II enzyme is an incompletely characterized mammalian protein that appears to be a membrane-bound metalloprotease localized in various tissues including muscle, cerebral cortex, and brain [6,8,9]. In contrast to the broad speci¢city of type I enzyme, type II enzyme is highly speci¢c for TRH. Both classes of the mammalian enzymes have been implicated in the regulation of neuropeptide activity, for example in the TRH pathway [10], and in the neurotensin system [11]. Although the role of bacterial enzymes still remains unclear, it has been proposed that PGP activity serves to reduce the toxicity of N-terminally blocked peptides [6], or plays a role in nutrient assimilation [1].
0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 1 8 5 - 6
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Fig. 1. Structure of pyroglutamyl peptidases I from B. amyloliquefaciens (1) and T. litoralis (2). Secondary structural elements are shown in a di¡erent color for each subunit. The subunits are named A to D. (1) BPGP: Cys68, Cys144 and Ser185, and (2) TPGP: Cys143 and Cys190 are drawn as ball-and-stick.
We reported the cloning of the PGP gene from Bacillus amyloliquefaciens (BPGP), characterization of its enzymatic properties [12], the three-dimensional structure of the enzyme (Fig. 1 (1)) [13], and the mechanism of substrate recognition [14].
Recently, Singleton et al. reported the crystal structure of the PGP from the hyperthermophilic archaeon Thermococcus litoralis (TPGP) [15]. TPGP showed enhanced thermal stability when compared with enzymes from mesophilic bacteria. TPGP is
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also a homotetramer, and its three-dimensional structure is similar to that of BPGP (Fig. 1 (2)). However, there were two disul¢de bonds between the subunits (A-ss-B, C-ss-D) in TPGP, but not in BPGP. This intersubunit disul¢de bond seems to be responsible for the thermal stability of the TPGP. We found that the introduction of the intersubunit disul¢de bond enhanced the thermal stability of BPGP without any change in its catalytic e¤ciency. 2. Materials and methods 2.1. Materials Restriction endonucleases and various DNA-modifying enzymes were obtained from New England Biolabs or Toyobo Biochemicals. Pyroglutamyl-Lnaphthylamide (pGlu-2NNap) and Fast Garnet GBC were from Sigma. The oligonucleotide primer for site-directed mutagenesis was synthesized by Amersham Pharmacia Biotech. The Mutan-Super Express Km kit and plasmid pKF18k were from Takara Shuzo. The ALF express AutoCycle sequencing kit and other reagents for DNA sequencing were obtained from Amersham Pharmacia Biotech. 2.2. Bacterial strains, plasmids, and medium Escherichia coli MV1184 was used for site-directed mutagenesis. E. coli DH1, JM83, and XLI-Blue were used as a host for expression. Plasmids (pUC18 and pKF18k) were used for expression and mutagenesis as a vector. Bacteria were grown in Luria^Bertani broth (LB broth). 2.3. Site-directed mutagenesis Site-directed mutagenesis was performed according to ODA (Oligonucleotide-directed Dual Amber)-LA PCR method using a Mutan-Super Express Km kit [16,17]. Oligonucleotide primer 5P-CCGAGCCTCTGCCTTGATCAC-3P was used to replace Ser185 with Cys. PCR was carried out in a 50 Wl reaction mixture containing 5 pmol selection primer, 5 pmol mutation
primer, 10 ng of template DNA, 5 Wl of 10ULA PCR Bu¡er II (Mg2 plus), 8 Wl of deoxynucleoside triphosphate (dNTP) mixture at 2.5 mM each, and 2.5 units of TaKaRa LA Taq DNA polymerase. Ampli¢cation involved 30 cycles of denaturation at 94³C for 1 min, annealing at 55³C for 1 min and extension at 72³C for 4 min. After PCR, the reaction mixture was allowed to cool at 4³C. The DNA in the reaction mixture was precipitated by adding 5 Wl of 3 M sodium acetate and 125 Wl of cold 100% ethanol. The precipitate was washed twice with cold 70% ethanol, dried and dissolved in 5 Wl of sterilized water. This DNA solution containing plasmid DNA was used to transform E. coli MV1184, and the transformants were selected on LB plates containing 50 Wg/ml kanamycin. To obtain the C68S/S185C mutant, we used the NruI site between C68 and S185, which is absent from pUC18 vector. The C68S fragment was obtained by digesting pBPG5Cys68Ser [12] with EcoRI and NruI. It was extracted from the agarose gel using the QIAEX II agarose gel extraction kit (Qiagen), and substituted for the same fragment from pBPG5Ser185Cys to produce C68S/S185C double mutant. Mutation was con¢rmed by sequence analysis with the dideoxy chain termination method. Double-stranded plasmids as templates and pUC18speci¢c primers were used. The dideoxy sequencing reactions were performed using an AutoCycle sequencing kit. DNA fragments containing the mutation were prepared by digesting the respective plasmids with EcoRI and HindIII. These fragments were inserted into the same site of pUC18, and then cloned plasmids were used to transform E. coli DH1 or E. coli JM83 for protein expression. 2.4. Puri¢cation of wild-type and mutant enzymes Wild-type and mutant BPGP were puri¢ed as previously described [12]. E. coli DH1 or E. coli JM83 cells harboring a mutant pgp gene plasmid were aerobically cultured in 250 ml of LB medium containing ampicillin at 37³C for 17 h. Cells were harvested by centrifugation (8000Ug; 20 min), washed and resuspended in 20 mM Tris^HCl bu¡er (pH 7.0). The cells were sonicated (ultrasonic disruptor UD200, Tomy) in 30 ml of the above bu¡er. The cell
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Fig. 2. Alignment of pyroglutamyl peptidase sequences. Homology was 34% between the enzymes. B.a, B. amyloliquefaciens; T.l, T. litoralis.
debris was eliminated by centrifugation. The supernatant was fractionated with ammonium sulfate from 40% to 80% saturation, and resuspended in 20 mM Tris^HCl bu¡er (pH 7.0) containing 40% saturated ammonium sulfate. The solution was applied to a hydrophobic column of Toyopearl HW65C equilibrated with 20 mM Tris^HCl bu¡er (pH 7.0) containing 40% saturated ammonium sulfate. The column was washed with the above bu¡er, and the absorbed enzyme was eluted with decreasing linear gradient of ammonium sulfate, from 40% to 0% saturation, in 20 mM Tris^HCl bu¡er (pH 7.0). The active fractions were combined and concentrated by ultra¢ltration using an Amicon apparatus (PM-10) and desalted by dialyzing against 20 mM Tris^HCl bu¡er (pH 7.0) and Milli-Q water. The introduction of a disul¢de bond into the mutant enzyme was con¢rmed by 10% SDS^PAGE under reducing and oxidizing conditions.
scribed previously [12]. To 0.8 ml of 20 mM Tris^ HCl bu¡er (pH 7.0) were added 0.1 ml of 5.0 mM pGlu-2NNap dissolved in 10% dimethyl formamide and 0.1 ml of enzyme solution. After 5 min incubation at 37³C, the enzyme reaction was stopped by adding 0.5 ml of a Fast Garnet GBC (1 mg/ml) solution containing 10% Triton X-100 in 1 M acetate bu¡er (pH 4.0). The absorbance at 550 nm was measured after 15 min, using a Hitachi Model 100-20 or U-3000 spectrophotometer. One unit of activity was de¢ned as the amount of enzyme which released 1 Wmol of 2NNap per min under the above conditions. To determine the Km values, several concentrations of substrate, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 and 3.0 mM, were used. Lineweaver^Burk plots were made to calculate Km and apparent Vmax . To calculate kcat , the subunit molecular weight of wild-type BPGP, 23 286 was used.
2.5. Assay of enzyme activity
2.6. Thermal stability of the mutant enzymes
Pyroglutamyl peptidase activity was assayed spectrophotometrically by the release of L-naphthylamine due to enzymatic hydrolysis of pGlu-2NNap, as de-
The mutant enzymes and wild-type enzyme were preincubated at various temperatures for 30 min, and the remaining activity was measured at 37³C.
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3. Results and discussions 3.1. Intersubunit disul¢de bond formation From the comparison of the three-dimensional structure between BPGP [13] and TPGP [15], it was assumed that the higher thermal stability of TPGP arises from the presence of intersubunit disul¢de bridges. These two enzymes are homotetramers, and their three-dimensional structures are similar. However, an intersubunit disul¢de bond is formed from the Cys190 in TPGP, while it did not exist in BPGP. Amino acid sequence alignment around the Cys190 of TPGP is shown in Fig. 2. Since the Ser185 residue is corresponded in BPGP, we replaced the Ser185 with a Cys residue. The BPGP has two Cys residues (Cys68 and Cys144) per subunit molecule [12]. Cys144 formed the catalytic triad with Glu81 and His168, and the substitution of Cys144 with Ser resulted in a complete loss of activity [12,13]. However, the substitu-
Fig. 4. Thermal stability of the wild-type and mutant BPGPs. The enzyme (8.7 WM) in 20 mM Tris^HCl bu¡er (pH 7.0) was incubated at the indicated temperatures for 30 min, and the residual activities were assayed. a, wild-type; b, S185C; O, C68S/S185C.
tion of Cys68 with Ser did not a¡ect the activity at all [12]. To avoid the formation of the undesirable disul¢de bond between Cys68 and the replaced Cys185, in addition to the S185C mutant, the C68S/S185C double mutant was also constructed. Two mutant enzymes were constructed and could successfully be expressed in E. coli. Wild-type, S185C, and C68S/S185C mutant enzymes were similar with respect to expression levels and yields of puri¢cation (1.5 mg from 250 ml culture). Two mutant enzymes were analyzed by SDS^PAGE with and without 2-mercaptoethanol (2-ME). As shown in Fig. 3, the molecular mass of the wild-type enzyme was about 30 kDa with and without 2-ME. However, the molecular masses of both mutants were about 30 and 60 kDa, with and without 2-ME, respectively. These results suggested that the disul¢de bridge beTable 1 Kinetic constants of wild-type and mutant BPGPs Fig. 3. Con¢rmation of an intersubunit disul¢de bond by SDS^ PAGE. Lanes: 1 and 4, wild-type enzymes; 2 and 5, S185C mutant enzymes; 3 and 6, C68S/S185C mutant enzymes. Lanes 1^3 contained 1% 2-ME in the SDS loading bu¡er as a reducing agent, but in lanes 4^6 it was omitted.
Wild-type S185C C68S/S185C
Km (mM)
kcat (s31 )
kcat /Km (s31 mM31 )
0.25 0.18 0.16
785 770 641
3140 4278 4006
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Fig. 5. E¡ects of pH on the stability. The enzyme was preincubated for 30 min at 30³C and the indicated pH values, and the activity remaining was assayed. a, wild-type; b, S185C.
tween the substituted Cys185 bonded two subunits. Without 2-ME, a faster migrating band close to the 60 kDa band appeared. There is a possibility that cysteine at the active site also formed a disul¢de bridge in a dimer. 3.2. Kinetic parameters of mutant enzymes The kinetic constants, kcat and Km , of the enzymes were measured at 37³C using pGlu-2NNap as substrate. As shown in Table 1, the kcat /Km of the two mutant enzymes for pGlu-2NNap was similar to that of the wild-type. This result indicated that the introduction of the disul¢de bond did not a¡ect the catalytic e¤ciency of the enzyme. 3.3. Physicochemical properties The thermal stability of the wild-type and mutant enzymes was investigated in terms of the activity remaining after incubation at various temperatures. The mutant enzymes showed enhanced thermal stability compared to the wild-type (Fig. 4). Fifty percent remaining activity of the two mutant enzymes was attained at a temperature approx. 30³C higher than that of wild-type enzyme. However, the
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wild-type and mutant enzymes showed no di¡erence in thermal stability under reducing conditions with 1 mM 1,4-dithio-DL-threitol. Therefore, the enhanced thermal stability was due to the introduction of the intersubunit disul¢de bond. The stability of the mutant enzymes compared to the wild-type under various pH conditions was also investigated. Both types showed the same stability in the range 6^8, and retained more than 80% of maximal activity. The S185C mutant enzyme was more stable than the wild-type enzyme at pH 4 or 12 (Fig. 5). Disul¢de bonds can make considerable contributions to stability, an e¡ect mainly attributed to the decreased entropy of the denatured protein [18^20]. Many attempts have been made to increase protein stability by introducing `intramolecular' disul¢de bonds [19,21^24]. In the present study we succeeded to stabilize the BPGP by the introduction of `intermolecular' disul¢de bonds. This seems to be the ¢rst report that intersubunit disul¢de bonds can impart thermal stability in oligomeric enzymes. References [1] R.F. Doolittle, R.W. Armentrout, Biochemistry 7 (1968) 516^521. [2] A. Szewczuk, M. Mulczyk, Eur. J. Biochem. 8 (1969) 63^67. [3] D. Tsuru, K. Fujiwara, K. Kado, J. Biochem. 84 (1978) 467^ 476. [4] W.L. Taylor, J.E. Dixon, J. Biol. Chem. 253 (1978) 6934^ 6940. [5] D. Tsuru, K. Sakabe, T. Yoshimoto, K. Fujiwara, J. Pharmacobiodyn. 5 (1982) 859^868. [6] A.C. Awade, P. Cleuziat, T. Gonzales, J. Robert-Baudouy, Proteins 20 (1994) 34^51. [7] K. Fujiwara, R. Kobayashi, D. Tsuru, Biochim. Biophys. Acta 570 (1979) 140^148. [8] G. Czekay, K. Bauer, Biochem. J. 290 (1993) 921^926. [9] S. Wilk, E.K. Wilk, Neurochem. Int. 15 (1989) 81^90. [10] J. Boler, F. Enzmann, K. Folkers, C.Y. Bowers, A.V. Schally, Biochem. Biophys. Res. Commun. 37 (1969) 705^ 710. [11] T. Nakajima, T. Taminura, J.J. Pisano, Fed. Proc. 29 (1970) 282. [12] T. Yoshimoto, T. Shimoda, A. Kitazono, T. Kabashima, K. Ito, D. Tsuru, J. Biochem. 113 (1993) 67^73. [13] Y. Odagaki, A. Hayashi, K. Okada, K. Hirotsu, T. Kabashima, K. Ito, T. Yoshimoto, D. Tsuru, M. Sato, J. Clardy, Structure 7 (1999) 399^411. [14] K. Ito, T. Inoue, T. Takahashi, H-S. Huang, T. Esumi, S.
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[21] H. Takagi, T. Takahashi, H. Momose, M. Inouye, Y. Maeda, H. Matsuzawa, T. Ohta, J. Biol. Chem. 265 (1990) 6874^ 6878. [22] J.H. Ko, W.H. Jang, E.K. Kim, H.B. Lee, K.D. Park, J.H. Chung, O.J. Yoo, Biochem. Biophys. Res. Commun. 221 (1996) 631^635. [23] J. Mansfeld, G. Vriend, B.W. Dijkstra, O.R. Veltman, B. Van den Burg, G. Venema, R. Ulbrich-Hofmann, V.G. Eijsink, J. Biol. Chem. 272 (1997) 11152^11156. [24] H. Takagi, K. Hirai, M. Wada, S. Nakamori, J. Biochem. 128 (2000) 585^589.
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