The Mechanism of Substrate Recognition of Pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as Determined by X-ray Crystallography and Site-directed Mutagenesis*

Kiyoshi ItoDagger , Takahiko InoueDagger , Tomoyuki TakahashiDagger , Hua-Shan HuangDagger , Tomoyuki EsumiDagger , Susumi HatakeyamaDagger , Nobutada Tanaka§, Kazuo T. Nakamura§, and Tadashi YoshimotoDagger

From the Dagger  School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan and the § School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

Received for publication, December 27, 2000, and in revised form, February 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O···H-N:Inh and Gln71:N-H···OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The kcat value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (kcat/Km) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pyroglutamyl-peptidase I (PGP1-1, EC 3.4.19.3) is an aminopeptidase that is able to specifically remove the amino-terminal pyroglutamyl residue. PGP is widely distributed in bacteria, plants, and animals (1). Many biologically active peptides (thyrotropin-releasing hormone, luteinizing hormone-releasing hormone, neurotensin, etc.) and proteins have pyroglutamyl residues. The enzyme seems to be involved in the metabolism of these biological peptides and proteins. On the other hand, PGP-1 has been used in protein sequencing to unblock proteins and polypeptides with the amino-terminal pyroglutamyl residue prior to Edman degradation.

PGP-1 was first isolated from Bacillus amyloliquefaciens, and its enzymatic properties were characterized by us (2). We have also reported the cloning and sequencing of the enzyme gene (3). Genes have been cloned for the enzymes of Bacillus subtilis (4), Pseudomonas fluorescens (5), Staphylococcus aureus (6), and Streptococcus pyrogenes (7). By site-directed mutagenesis analysis, Cys-144 was estimated to be involved in the catalytic reaction (3). The enzyme was expressed in Escherichia coli and was crystallized. The three-dimensional structure of the enzyme was clarified by x-ray crystallography (8). The active site (a catalytic triad composed of Cys-144, His-168, and Glu-81) of each monomer was located inside the doughnut-shaped tetramer. A thermostable enzyme from Thermococcus litoralis was also studied by x-ray crystallography (9). However, the mechanism of substrate specificity was unclear.

To elucidate the catalytic mechanism and biological significance of the enzyme, several specific inhibitors have been synthesized (10-13). In this study, we analyzed the mechanism of substrate recognition for pyroglutamyl residue by x-ray crystallography of the enzyme-inhibitor complex and by site-directed mutagenesis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Restriction endonucleases, other modification enzymes, and a Mutan-Super Express Km kit were purchased from TaKaRa Shuzo Co. The oligonucleotide primers containing the desired changes for the mutants were synthesized by Amersham Pharmacia Biotech. The AutoCycle sequencing kit and other reagents used for sequencing were also obtained from Amersham Pharmacia Biotech. Polyethylene glycol, which was used for crystallization with a mean molecular weight of 4000, was purchased from Nacalai Tesque. 5-hydroxymethyl-2-pyrrolidinone was obtained from Aldrich.

Preparation of Pyroglutaminal (5-Oxo-pyrrolidine-2-carbaldehyde)-- Pyroglutaminal was synthesized by the one step method of Frigerio et al. (14). To a stirred solution of 5-hydroxymethyl-2-pyrrolidinone (1.03 g, 8.96 mmol) in Me2SO (10 ml) at room temperature was added o-iodoxybenzoic acid (3.03 g, 10.9 mmol). After 2 h, most of the Me2SO was removed under reduced pressure, and the residue was purified twice by column chromatography (SiO2 (100 g), tetrahydrofuran; then SiO2 (30 g), ethylacetate/isopropanol (3/1)) to give 5-oxo-pyrrolidine-2-carbaldehyde (337.2 mg, 33%) as a colorless oil.

Bacterial Strains, Plasmids, and Media-- E. coli MV1184 (ara, Delta  (lac-proAB), rpsL, thi (Phi 80 lacZDelta M15), Delta  (srl-recA) 306::Tn10 (tetr)/F' (traD36, proAB+, lacIq, lacZDelta M15)) was used for site-directed mutagenesis. E. coli DH1 (supE44, hsdR17, recA1, endA1, gyrA96, thi-1, relA1) and DH5alpha (F-, Phi 80dlacZDelta M15, Delta (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rk-mk+), phoA, supE44, lambda -, thi-1, gryA96, relA1) were used for expression. Plasmids pUC18 and pKF18k were used for expression and mutagenesis.

Construction of the Site-directed Mutant Gene-- Oligonucleotide-directed dual amber long and accurate polymerase chain reaction method was used to construct six mutants: F10A, F10Y, F13A, F13Y, F142A, and F142Y. A Mutan-Super Express Km kit based on the oligonucleotide-directed dual amber method was used to make all mutants. The pgp gene on a 0.9-kilobase EcoRI-HindIII fragment was ligated into pKF18k, yielding pKF18-PG, which was used as a template. The primers for mutation are shown in Table I. Polymerase chain reaction was carried out in 50 µl of a reaction mixture containing 5 pmol of selection primer, 5 pmol of mutagenized primer, 10 ng of template DNA, 5 µl of 10 × long and accurate polymerase chain reaction buffer II (Mg2+ plus), 8 µl of a dNTP mixture (2.5 mM each), and 2.5 units of TaKaRa long and accurate Taq DNA polymerase. Amplification consisted of 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 3 min. After the polymerase chain reaction, the reaction mixture was allowed to cool to 4 °C.

                              
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Table I
Primers for mutagenesis

The DNA in the reaction mixture was precipitated by adding 25 µl of 4 M ammonium acetate and 100 µl of cold 100% ethanol. The precipitate was washed twice with 70% ethanol, dried, and dissolved in 5 µl of buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA).

This DNA solution was used to transform E. coli MV1184, and transformants were selected on LB plates containing 30 µg/ml kanamycin. Mutations were confirmed by digesting the respective plasmids with EcoRI and HindIII, and the successful mutations were used to transform E. coli DH1 or DH5alpha for expression.

Purification of the Mutant Enzymes-- E. coli transformed by mutant plasmids were aerobically cultured in 12 liters of N-broth containing ampicillin (50 mg/liter) at 37 °C for 17 h in a jar fermentor (New Brunswick Co.). The harvested cells were washed with a 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM EDTA and resuspended in the same buffer. The cells were disrupted with glass beads in a Dyno-Mill, and the suspension was centrifuged to remove cell debris. To the supernatant were added protamine sulfate drops at 18 mg per g of wet cells to remove chromosomes and viscous materials. The mixture was kept for 30 min and then centrifuged. The supernatant was fractionated with ammonium sulfate at 40-80% saturation. The precipitate was dissolved in a small volume of 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM EDTA and 40% saturated ammonium sulfate, and the suspension was dialyzed against the same buffer.

The resultant clear solution was applied to a hydrophobic column of Toyopearl HW-65C equilibrated with the above buffer. The column was washed with the buffer, and the adsorbed enzyme was eluted with a decreasing linear gradient of ammonium sulfate, from 40 to 0% saturation, in 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM EDTA. The active fraction of the eluate was combined and concentrated with ammonium sulfate at 80% saturation. The resultant precipitate was desalted by dialyzing against 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM EDTA and concentrated by ultrafiltration. The resultant concentrated solution was dialyzed against 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM EDTA and ammonium sulfate at 40%. Many small needle crystals appeared after 2-4 days. The crystals were dialyzed against H2O and concentrated by ultrafiltration (3).

Enzyme Activity Assay and Kinetic Studies-- The activity of pyroglutamyl-peptidase was assayed as described by Yoshimoto et al. (3). To 0.8 ml of 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM EDTA were added 5.0 mM L-pyroglutamyl-beta -naphthylamide dissolved in 10% dimethyl formamide and 0.1 ml of enzyme solution. After 5 min of incubation at 37 °C, the reaction was stopped by adding 0.5 ml of Fast Garnet GBC (1 mg/ml) solution containing 10% Triton X-100 in 1 M acetate buffer (pH 4.0). The absorbance at 550 nm was measured after 15 min. One unit of the activity was defined as the amount of enzyme that released 1 µmol of beta -naphthylamine per min under the above conditions.

To determine the Km values, the concentration of the substrate was varied. Lineweaver-Burk plots were used to calculate Km and apparent Vmax. To calculate kcat, the subunit molecular weight of wild-type PGP-1 (23,286) was used.

Crystallization-- Crystallization of the wild-type PGP-1 was attempted using the hanging drop vapor diffusion method. The crystallization was performed at pH 6.5, in 0.05 M sodium cacodylate, KH2PO4 buffer with 0.1 M magnesium acetate, using 10% (w/v) polyethylene glycol 4000 as the precipitant. The protein concentration was 7 mg/ml. All mutants were crystallized under the same conditions used for the wild type. Crystals of the F142A mutant were obtained using the hanging drop vapor diffusion method with microseeding and macroseeding under the conditions (pH 6.4) used for the crystallization of the wild-type PGP-1. To obtain crystals of the enzyme-inhibitor complex, the crystals were soaked in the standard solution (0.05 M sodium cacodylate, KH2PO4 buffer with 0.1 M magnesium acetate and 15% (w/v) polyethylene glycol 4000) containing 100 mM pyroglutaminal for 1 week at 20 °C.

X-ray Data Collection and Processing-- Before x-ray data were collected, crystals of mutants were transferred into 0.6 ml of fresh standard solution and incubated for 2-3 days at 20 °C. Intensity data for the inhibitor complex and the F142A mutant derivative were collected at 20 °C on a Rigaku R-AXISIIc area detector with graphite-monochromated CuKalpha radiation, which was generated by a Rigaku RU200 rotating anode x-ray generator (operated at 40 kV and 90 mA). The program PROCESS installed in the R-AXISIIc system was used for data collection and processing. The completeness of the F142A mutant data set was 82.3% in the resolution range between 15.0 and 2.06 Å, whereas that of the inhibitor complex data set was 83.7% between 15.0 and 2.06 Å.

Model Building and Structure Refinement-- The crystal structure of the wild type has been determined already using the single isomorphous replacement method based on a p-chloromercuribenzoate derivative. The crystal structures of the F142A mutant and the inhibitor complex were solved using the molecular replacement method with the coordinates of the wild-type structure as a model and were refined by the program X-PLOR (15). The mutated residues were fitted to a 2Fo - Fc map using the program TURBO-FRODO (16).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

X-ray Crystallographic Analysis of the Enzyme-Inhibitor Structure-- Pyroglutaminal was synthesized from 5-hydroxymethyl-2-pyrrolidinone in one step (Fig. 1). To study the structure of the inhibitor-bound enzyme, wild-type enzyme was crystallized in the absence of inhibitor as described earlier (8). Then, the crystals were soaked in the standard buffer containing 100 mM inhibitor. Data were collected for crystals soaked for 1, 2, 4, and 7 days. The best diffraction data were obtained from the crystal soaked for 1 week. The crystal belongs to a monoclinic space group P21, the same as the wild type. Cell dimensions of the enzyme-inhibitor complex were as follows: a = 78.51 Å; b = 80.36 Å; c = 69.25 Å; and beta  = 92.99°. The crystal structure of the complex was solved using the difference Fourier method with the coordinates of the wild type as the starting model (10). The model was refined using the program X-PLOR for energy restraints and optional dynamics at high temperature. The refinement parameters are summarized in Table II.


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Fig. 1.   Synthesis of pyroglutaminal (5-oxo-pyrrolidine-2-carbaldehyde). DMSO, dimethyl sulfoxide.

                              
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Table II
Crysrallographic and refinement parameters of the inhibitor complex and the mutant enzyme
Values in parentheses refer to the highest resolution shell.

Fig. 2 shows the 2Fo - Fc electron density map for the pyroglutaminal directly connected to the active cysteine 144. To exclude a bias toward any type of conceived model, the structure was specifically refined in the absence of the inhibitor molecule before the map calculation. The well shaped electron density in the map clearly delineates the conformation of the inhibitor. The structure around the active site is shown in Fig. 3. The nucleophile of Cys-144 attacks the aldehyde carbon atom, resulting in the formation of a covalent thiohemiacetal. Two hydrogen bonds are present between the inhibitor and the enzyme.


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Fig. 2.   Stereoscopic drawing of the electron density map around the catalytic cysteine residue of the pyroglutamyl-peptidase-pyroglutaminal (PGL) complex. The inhibitor molecule (at the center of the figure) is superimposed on the 2Fo - Fc annealed omit electron map contoured at 1.0 sigma  using TURBO-FRODO (16)


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Fig. 3.   Stereoscopic drawing of the binding structure of the pyroglutaminal. Possible hydrogen bonds between the enzyme and the inhibitor are shown with black lines. The van der Waals surface of the inhibitor is shown by dots using TURBO-FRODO (16)

Site-directed Mutagenesis-- In addition to the hydrogen bonds, the crystal structure of the complex revealed a hydrophobic pocket consisting of Phe-10, Phe-13, and Phe-142. These residues were conserved in the multiple alignment of seven pyroglutamyl-peptidase sequences. These three phenylalanines were selected for further study by site-directed mutagenesis. In the present study, they were changed to Tyr and Ala (F10Y, F10A, F13Y, F13A, F142Y, and F142A). All substitutions and codon changes are summarized in Table I. These mutated DNAs were sequenced to confirm the presence of correct codon substitutions without other alterations.

Expression and Purification of the Mutant Enzymes-- Mutant pgp genes were expressed under the control of the lac promoter on the plasmid pUC18. SDS-polyacrylamide gel electrophoresis showed that all mutants were overexpressed under the standard conditions used for wild-type enzyme production. All enzymes were purified from cell-free extracts of the respective transformants as described under "Experimental Procedures."

Because the mutant enzyme F10A was precipitated with a low ammonium sulfate concentration, it could not be purified. All mutants, except F10A, showed similar chromatographic behavior to that of the wild type (3). The purity of the mutant enzymes was analyzed by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 4, a single band corresponding to the migration of wild-type enzyme was obtained for each mutant.


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Fig. 4.   SDS-polyacrylamide gel electrophoresis of pyroglutamyl-peptidase mutants purified with Toyopearl HW65C. Lane M, marker; lane 1, F10Y; lane 2, F13Y; lane 3, F142Y; lane 4, F13A; lane 5, F142A.

Kinetic Analysis of the Mutant Enzymes-- The kinetic parameters of each mutant obtained using L-pyroglutamyl-beta -naphthylamide as substrate are summarized in Table III. Among the tyrosine substitution mutants, F13Y and F142Y had kinetic parameters similar to those of the wild type. However, the Km value and the kcat obtained for the F10Y mutant were 3.6-fold higher and 5.8-fold lower, respectively, than the wild-type values, and thus kcat/Km of F10Y decreased more than 20-fold. When phenylalanines were replaced with the rather small amino acid alanine, more remarkable changes in kinetic parameters were observed. The kcat/Km of the F13A and F142A mutants decreased more than 1000-fold. These drastic changes were mainly due to the decrease in the kcat value of the mutants. Kinetic parameters for F10A were not assessed, because the mutant could not be purified, as described above. The mutant seemed to be severely destabilized and suffered a major structural change.

                              
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Table III
Kinetic constants for wild type and mutants of PGP-1

X-ray Crystallographic Analysis of the Mutant F142A-- After a 1-week incubation in the standard buffer, small crystals appeared for F10Y, F142Y, and F142A. Using the seeding technique, crystals of the F142A mutant and the enzyme-inhibitor complex were grown to ~0.3-0.5 mm. Both crystals belong to a monoclinic space group, P21. Cell dimensions were as follows: a = 78.47 Å; b = 80.95 Å; c = 69.22 Å; and beta  = 92.74°.

The mutant model was refined using the program X-PLOR for energy restraints and optional dynamics at high temperature. Refinement parameters are summarized in Table II.

The overall structure of F142A was similar to that of the wild type, suggesting that the observed changes in kinetic parameters were due to the local environment, especially in the substrate-recognizing hydrophobic pocket. The changes in electron density associated with the amino acid substitution were inspected with omit maps. Fig. 5 shows the 2Fo - Fc electron density map for residue 142 and the Ala-142 molecular model. There is no electron density corresponding to the benzene ring at position 142. This clearly proved the presence of an alanine residue at position 142. 


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Fig. 5.   The 2Fo - Fc electron density map around position 142 of the F142A mutant. The map was contoured at 1.0 sigma  using TURBO-FRODO (16).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The study of the inhibitor-complex structure provides insight into the inhibitor-protein interactions that contribute to the binding between the inhibitor and the enzyme. The primary binding force is likely to be two hydrogen bonds between the inhibitor and the main chain of the enzyme. These hydrogen bonds help to orient the pyroglutamyl residue for nucleophilic attack by Cys-144 at the active site and to stabilize the complex. Van der Waals interaction between the pyrrolidone ring and the hydrophobic pocket, which consists of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143, also seems to be essential. Interestingly, the benzene rings of three phenylalanines of PGP-1 and the pyrrolidone ring of the inhibitor are almost parallel, and the pyrrolidone ring was held by Phe-10, Phe-13, and Phe-142 (Fig. 3). Fig. 6 provides a direct view of the large movement of Phe-13 and Gln-71 in the inhibitor-complex structure. It is remarkable that only Phe-13 and Gln-71 serve as an induced fit mechanism about which the hydrophobic pocket closes.


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Fig. 6.   Superposition around the hydrophobic pocket of the wild type (blue) and the wild type-pyroglutaminal complex (brown). Pyroglutaminal (PGL) is shown in pink.

Recently, we have reported the crystal structure of prolyl aminopeptidase from Serratia marcescens (17). Because proline and pyroglutamic acid have structures in common with the pyrrolidone ring, a similar substrate recognition mechanism was expected. In the prolyl aminopeptidase from S. marcescens, the hydrophobic pocket (Ala-270, Phe-139, Phe-236, Trp-114, Trp-148, and Cys-271) was located near the catalytic triad (Ser-113, His-296, and Asp-268). The role of one of the residues composing the hydrophobic pocket was elucidated by comparing prolyl aminopeptidase structure. The crystal structure of prolyl oligopeptidase complexed with benzyloxycarbonyl-Pro-prolinal was clarified by Fulop et al. (18). The S1 specificity pocket ensures a hydrophobic environment (Trp-595, Phe-476, Val-644, Val-580, and Tyr-599) and a snug fit for the proline residue, and the specificity for proline residue was enhanced by the stacking between the indole ring of Trp-595 and the pyrrolidine ring of the substrate proline residue (distance, 3.4 to ~3.8 Å). From the superposition of both active sites, Phe-139 of prolyl aminopeptidase was estimated to have the same role as Trp-595 of prolyl endopeptidase (19). However, no remarkable hydrogen bonding between the proline residue of the inhibitor and prolyl oligopeptidase was found. This suggests that these two enzymes use somewhat similar but distinct mechanisms for substrate recognition, although both have a hydrophobic pocket, in which an aromatic residue plays some role in binding the pyrrolidone ring.

To elucidate whether the conserved phenylalanine residues in PGP-1 play a role in substrate recognition, we used the site-directed mutagenesis method. First, three phenylalanine residues were replaced with tyrosines, which are not markedly different in size. The F10Y mutant showed a decrease in kcat, whereas the two other mutants, F13Y and F142Y, did not exhibit significant changes in their kinetic parameters compared with the wild-type enzyme. The decrease in kcat of F10Y mutant seems to be due to steric hindrance and a decrease in hydrophobicity.

Phenylalanine was replaced with alanine, which is considerably different in both structure and size. SDS-polyacrylamide gel electrophoresis analysis showed that all mutants were expressed at similar levels. However, the F10A mutant showed very little activity and was precipitated by lower concentrations of ammonium sulfate than those used for the other mutants. Because a marked change in protein structure apparently occurred in F10A, Phe-10 would be a key residue for enzyme structure. The overall structure of F10Y changed little, because the mutant formed crystals under the same conditions used for the wild-type enzyme. Nevertheless, even a conservative substitution of tyrosine for Phe-10 caused a 20-fold decrease in catalytic efficiency (kcat/Km), suggesting that Phe-10 is important for substrate recognition. Other mutants, F13A and F142A, were purified to homogeneity using the standard method. The catalytic efficiencies (kcat/Km) of F13A and F142A decreased more than 1000-fold compared with that of the wild-type enzyme. The crystal structure of F142A clearly showed that Phe-142 was replaced with Ala. The superimposition of active sites of F142A and the wild-type enzyme shows no significant change in the main chain; Gln-71 can hydrogen bond to the carbonyl oxygen of the pyrrolidone ring. Cys-144 and His-168, which compose the catalytic triad, remain in almost the same position. However, minor shifts of Phe-13 and Phe-142 were observed as a result of the removal of the benzene ring of Phe-142 (data not shown).

In conclusion, the enzymatic recognition of the pyrrolidone ring of pyroglutamic acid is achieved by two hydrogen bonds between the inhibitor and the main chain located at the side of the cavity. On the other side of the cavity, three phenylalanine residues compose the hydrophobic pocket, the benzene rings of three phenylalanines of PGP-1 and the pyrrolidone ring of the inhibitor are almost parallel, and the substrate pyrrolidone ring is fixed in the pocket by those residues. Phe-10 plays an essential role, and Phe-13 serves as an induced fit mechanism, whereas Phe-142 provides the hydrophobic properties. As shown in Fig. 7, Phe-10 was conserved in all the enzymes reported to date; however, Phe-13 and Phe-142 were replaced with Tyr in some enzymes. These results also support a role for these phenylalanine residues.


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Fig. 7.   Alignment of proglutamyl-peptidase I sequences. Conserved Phe-10, Phe-13, and Phe-142 residues are boxed. B.a, B. amyloliquefaciens (3); B.s, B. subtilis (4); S.p, S. pyrogenes (7); L.l, Lactococcus lactis (20); S.a, S. aureus (6); Ps.f, P. fluorescens (5); S.c, Streptomyces coelicolor (EMBL/GenBankTM/DDBJ Data Bank accession number AL121596, submitted by L. Murphy and D. Harris (1999)); Py.h, Pyrococcus horikoshii (22); Py.f, Pyrococcus furiosus (21); T.l, T. litoralis (9); M.b, Mycobacterium bovis (EMBL/GenBankTM/DDBJ Data Bank accession number U91845, submitted by J. K. Kim and Y. K. Choe (1997)).


    Aknowledgements

We thank Dr. T. Kabashima and N. Ohta for assistance and helpful discussions.

    FOOTNOTES

* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 95-847-1111; Fax: 95-843-2444; E-mail: t-yoshimoto@cc.nagasaki-u.ac.jp.

Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M011724200

    ABBREVIATIONS

The abbreviation used is: PGP, pyroglutamyl-peptidase..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Robert-Baudouy, J., and Thierry, G. (1998) in Handbook of Proteolytic Enzymes (Barrett, A. J., Rawlings, N. D., and Woessner, J. F., eds) pp. 791-795
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