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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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,
(lac-proAB), rpsL,
thi (
80 lacZ
M15),
(srl-recA) 306::Tn10
(tetr)/F' (traD36,
proAB+, lacIq,
lacZ
M15)) was used for site-directed mutagenesis.
E. coli DH1 (supE44, hsdR17,
recA1, endA1, gyrA96,
thi-1, relA1) and DH5
(F
,
80dlacZ
M15,
(lacZYA-argF) U169, deoR,
recA1, endA1, hsdR17 (rk
mk+), phoA,
supE44, 
, 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.
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
DH5
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-
-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
-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 CuK
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).
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RESULTS |
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
= 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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Table II
Crysrallographic and refinement parameters of the inhibitor complex and
the mutant enzyme
Values in parentheses refer to the highest resolution shell.
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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 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)
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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.
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Kinetic Analysis of the Mutant Enzymes--
The kinetic parameters
of each mutant obtained using
L-pyroglutamyl-
-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.
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
= 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 using
TURBO-FRODO (16).
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DISCUSSION |
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.
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