Acylamino Acid-releasing Enzyme from the Thermophilic Archaeon
Pyrococcus horikoshii*
Kazuhiko
Ishikawa
§,
Hiroyasu
Ishida
,
Yoshinori
Koyama
,
Yutaka
Kawarabayasi
¶,
Jun-ichi
Kawahara
,
Eriko
Matsui
, and
Ikuo
Matsui
From the
National Institute of Bioscience and
Human-Technology and the
National Institute of Materials and
Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305, and the
¶ National Institute of Technology and Evaluation, Nishihara,
Shibuyaku, Tokyo 151, Japan.
 |
ABSTRACT |
When the genome of the thermophilic archaeon
Pyrococcus horikoshii was sequenced, a gene homologous to
the mammalian gene for an acylamino acid-releasing enzyme (EC 3.4.19.1)
was found in which the enzyme's proposed active residues were
conserved. The P. horikoshii gene comprised an open reading
frame of 1,896 base pairs with an ATG initiation codon and a TAG
termination codon, encoding a 72,390-Da protein of 632 amino acid
residues. This gene was overexpressed in Escherichia coli
with the pET vector system, and the resulting enzyme showed the
anticipated amino-terminal sequence and high hydrolytic activity for
acylpeptides. This enzyme was concluded to be the first acylamino
acid-releasing enzyme from an organism other than a eukaryotic cell.
The existence of the enzyme in archaea suggests that the mechanisms of
protein degradation or initiation of protein synthesis or both in
archaea may be similar to those in eukaryotes. The enzyme was stable at 90 °C, with its optimum temperature over 90 °C. The specific
activity of the enzyme increased 7-14-fold with heat treatment,
suggesting the modification of the enzyme's structure for optimal
hydrolytic activity by heating. This enzyme is expected to be useful
for the removal of N
-acylated
residues in short peptide sequence analysis at high temperatures.
 |
INTRODUCTION |
The acylamino acid-releasing enzyme
(AARE)1 catalyzes the
NH2-terminal hydrolysis of
N
-acylpeptides to release
N
-acylated amino acids (1). AARE has
been used for removal of N
-acylated
residues in protein sequence analysis. Until now, AARE has been
isolated only from eukaryotic cells (1- 4) and classified as its own
serine protease subfamily (5, 6). The physiological role of the enzyme
is not clear, although it has been suggested that it affects the
processing or sorting of proteins (7, 8) in eukaryotic cells. From
eukaryotic cells, some AARE genes have already been cloned (9, 10).
However, the production and expression of AARE from these genes within
Escherichia coli have not been carried out.
Pyrococcus horikoshii (OT3) is one of the thermophilic
archaea collected from a volcanic vent in the Okinawa trough (11). The
optimum growth temperature of this archaeon ranges from 90 to
105 °C. Most of the proteins from P. horikoshii are
thought to be thermostable and active at high temperature. The size of its genome is about 2 Mb, and the guanine-cytosine content is relatively low. At the National Institute of Technology and Evaluation (Tokyo, Japan), sequencing of this genome is in progress (11). From the
genome sequencing in P. horikoshii we found a gene that had
some homology with a gene for AARE from pig liver (9, 12). Therefore,
we cloned the gene from P. horikoshii and attempted to
express the enzyme in E. coli and examine the
characteristics of the expressed enzyme.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The host E. coli BL21(DE3) and the
vector pET11a were obtained from Novagen (Madison, WI). The
Pfu DNA polymerase, restriction enzymes, and ligation kit
were purchased from Takara Shuzo (Otsu, Shiga, Japan). The
N
-acetylamino acid p-nitroanilide
derivatives (Ac-amino acid-pNA) were purchased from Sigma
(St. Louis, MO) and Bachem (Bubendorf, Switzerland). The acylpeptides
Ac-Ala-Ala, Ac-Met-Ala, f-Met-Ala, f-Met-Ala-Ser, f-Met-Leu-Gly, and
f-Ala-Ala-Ala were also from Bachem. The other acylpeptides
Ac-Met-Ala-Ala-Ala-Ala-Ala, Ac-Ala-Ala-Ala-Ala, Ac-Ala-Ala-Ala-Ala-Ala-Ala, f-Met-Ala-Ala-Ala-Ala-Ala, and
f-Ala-Ala-Ala-Ala-Ala-Ala were purchased from Peptide Institute Inc.
(Minou, Osaka, Japan).
-Melanocyte-stimulating hormone (
-MSH) was
purchased from Funakoshi (Tokyo, Japan). The synthesis of DNA primers
and the sequencing of proteins were performed by the custom service
center of Takara Shuzo. The DNA sequencing was carried out with ABI
model 373 sequencer (Perkin-Elmer, Applied Biosystems Div., Foster
City, CA). All other chemicals were of the highest reagent grade
commercially available.
Cloning and Expression of the Gene--
The genome of P. horikoshii was sequenced by the method of Kaneko et al.
(13). The gene that was homologous to the mammalian gene for AARE was
found by BLAST search (14). The gene was amplified by the polymerase
chain reactionmethod using two primers with unique restriction sites.
Amplification of the gene by polymerase chain reaction was carried out
at 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 3 min, for
35 cycles using Pfu DNA polymerase. The amplified gene was
hydrolyzed by the restriction enzymes and inserted in pET11a cut by the
same restriction enzymes. The amplified gene was expressed using the
pET11a vector system in the host E. coli BL21(DE3) according
to the manufacturer's instructions. The host E. coli
BL21(DE3) was transformed by the constructed plasmid. The transformant
cell was grown in 2YT medium (1% yeast extract, 1.6% tryptone, and
0.5% NaCl) containing ampicillin (100 µg/ml) at 37 °C. After
incubation with shaking at 37 °C until the
A600 reached 0.6-1.0, the induction
was carried out by adding isopropyl
-D-thiogalactopyranoside at a final concentration of 1 mM and shaking for 4 h at 37 °C. The concentration
of the enzyme was determined with Coomassie protein assay reagent
(Pierce Chemical Company, Rockford, IL) using bovine serum albumin as
the standard protein.
Purification of the Enzyme--
After induction, the
transformant cells were harvested by centrifugation and disrupted with
oxide aluminum in 50 mM Tris-HCl buffer (pH 8.0) containing
0.6 M NaCl. After incubation with DNase I (from bovine
pancreas; Sigma) for 30 min at room temperature, the crude extract was
heated at 85 °C for 30 min. The supernatant obtained by
centrifugation was dialyzed against 50 mM Tris-HCl buffer
(pH 8.0). The dialyzed sample was loaded on a HiTrap Q column
(Pharmacia, Uppsala, Sweden). The column was washed with 50 mM Tris-HCl buffer (pH 8.0) and eluted with a linear
gradient (0-1.0 M NaCl in the same buffer). The fractions
that showed protein of a similar molecular mass(70 kDa, SDS-PAGE)
calculated from the amino acid sequence were concentrated by a
Centricon 10 filter (Amicon Inc., Beverly, MA). The concentrated
material was loaded on a HiLoad Superdex 200 column (Pharmacia) and
eluted with 100 mM Tris-HCl buffer (pH 8.0) containing 1.0 M NaCl. The fractions demonstrating only one protein band
with a molecular mass of 70 kDa by SDS-PAGE were collected and used for
the detailed characterizations of the enzyme.
Molecular Weight Determination--
The molecular weight of the
enzyme was determined by SDS-PAGE performed on a 4-15% gradient gel
in the Phast System (Pharmacia). Protein bands were visualized by
staining with Coomassie Brilliant Blue.
The molecular weight was also determined by high performance liquid
chromatography (HPLC) and light-scattering photometry. The HPLC was
performed on a Superdex 200 column (Pharmacia), and the elution was
carried out using 100 mM Tris-HCl buffer (pH 8.0) containing 1.0 M NaCl at 1.5 ml/min at room temperature.
The eluted protein was detected by its absorbance at 280 nm. The
light-scattering photometer was conducted at room temperature with a
DLS-700S light-scattering photometry (Otsuka Denshi, Shiga, Japan)
calibrated with benzene (15) at 633 nm and analyzed by the method of
Kamata and Nakahara (16). Optical clarification was performed with
polyvinylidene fluoride filters. The specific refractive index
increment (dn/dc) was obtained with a KMX-16 refractometer (Chromatix
Inc., Sunnyvale, CA at the same wavelength, calibrated with NaCl
solution.
Enzyme Assay--
The activity of the enzyme was determined
using Ac-amino acid-pNA and acylpeptides. The enzyme was
incubated at 85 °C with the substrates in 50 mM sodium
acetate buffer (pH 5.4) containing 0.6 M NaCl and 5%
N, N-dimethylformamide (DMF), and the released products were measured. The activity toward the Ac-amino
acid-pNAs was calculated using the absorption coefficient
406 = 9.91 mM
1 of
pNA released (17). The activity toward the acylpeptides was
measured by the detection of the exposed
-NH2 group with the cadmium-ninhydrin colorimetric method (18). The analysis of the
products from the peptides was performed by HPLC on an ODS-80Ts column
(4.6-mm inner diameter × 25 cm) containing TSK gel (Tosoh, Tokyo,
Japan). The flow rate was 0.7 ml/min with 95% water, 5% acetonitrile,
and 0.1% trifluoroacetic acid. The activity toward
-MSH was
examined by a PSQ-1 protein sequencer (Shimazu, Kyoto, Japan) at the
custom service center of Takara Shuzo.
1 unit of activity corresponds to the amount of enzyme which catalyzes
the hydrolysis of 1 µmol of substrate/min.
Measurement of Thermostability--
Thermostability of the
enzyme was measured by the circular dichroism (CD) and the differential
scanning calorimetry (DSC).
CD was measured with a CD spectrometer (model 62A DS) (Aviv Instrument,
Lakewood, NJ) utilizing a 5.0-mm path length quartz cell in the far UV
region. The scan rate of the temperature was 1 K/min. The measurement
was carried out in 50 mM sodium acetate buffer (pH 5.4)
containing 0.6 M NaCl.
The experiments of DSC were performed in a model DSC5100 calorimeter
(Calorimetry Sciences Corp., Provo, UT). A scan rate of 1 K/min was
used throughout. Before measurement, the sample was dialyzed against 50 mM sodium acetate buffer (pH 5.4) containing 0.6 M NaCl and degassed with an aspirator for 15 min.
Instrument base lines were established with both cells filled with
dialysate; the reference cell remained filled with dialysate during the
protein scans.
 |
RESULTS AND DISCUSSION |
Expression of the Enzyme--
In the genome sequenced from
P. horikoshii, we found a gene that contained 1,896 base
pairs and showed about 20% identity with the AARE gene from pig liver
(Fig. 1) (12). The open reading frame was
preceded by AT-rich regions in which a putative ribosome binding site
GGTGAT at position
4 and a putative promoter consensus TTATAT at
position
33 from ATG initiation site were found. This consensus
resembles the eukaryotic TATA box and has been confirmed to be the
archaeal consensus sequence TT(A/T)(T/A)AX, as determined by
analysis of more than 80 archaeal promoters (19). The protein encoded
consists of 632 amino acids, making it smaller than AARE (732 amino
acids) from a mammal. However, the proposed active residues (Ser, Asp,
and His) of AARE, called "catalytic triad residues" (5, 6, 12),
were conserved (Fig. 1). Furthermore, the sequence homology in the Ser,
Asp, and His regions (6) of a new family of serine-type peptidases (5)
was also observed in this protein. The residues Tyr-492 in the Ser
region and Glu-602 in the His region of this protein are not conserved
in AARE, but dipeptidyl peptidase (6). These results suggest that this
protein, dipeptidyl peptidase, and AARE might be evolutionally
related.

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Fig. 1.
Comparison of AARE sequences. Sequence
alignment was performed by a Genetyx-Mac program (Software Development
Co., Ltd., Tokyo, Japan). The sequences have been aligned with
dashes indicating gaps. Conserved residues between two
enzymes are marked with an asterisk (*). Putative active
residues (Ser-491, Asp-572, and His-604) are marked +.
Abbreviations: P. horikoshi, AARE from P. horikoshii.; Pig, AARE from pig liver.
|
|
The gene was amplified by polymerase chain reaction using two primers.
The upper primer (5'-TTTTGAATTCTTACATATGGGCAAGGGGCTTTCA-3') contained an NdeI I site (underlined), and the lower primer
(5'-TTTTGGTACCTTT GGATCC TAAGGGTTTAGCTATCCTTT-3') contained
a BamHI site (underlined). The amplified gene was inserted
in pET11a, and BL21(DE3) was transformed by the constructed plasmid.
After induction for 4 h at 37 °C, 50 mg of the thermostable
70-kDa protein (as determined by SDS-PAGE) was purified from 2 liters
of culture medium. The result of densitometer (data not shown) for
SDS-PAGE (Fig. 2) indicated that the
purity was about 99%. The purified protein (0.05 mg) in solution was spotted on an Immobilon polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) and sequenced by a PSQ-1 protein sequencer (Shimazu) at the custom service center of Takara Shuzo. By sequence analysis, the first 20 amino acid residues of the NH2
terminus except the NH2-terminal Met were detected. The
NH2-terminal sequence was identical to that anticipated
from the nucleotide sequence. The extra f-Met residue at the
NH2 terminus of the nascent polypeptide, encoded by the
initiation codon, was not detected. This shows that the Gly residue
neighboring the starting f-Met residue has a small radius of gyration
which is essential for the removal of the f-Met residue to yield the
mature enzyme (20), and the soluble protein was processed correctly.
The high yield of the recombinant protein indicates the very efficient
post-translation of the P. horikoshii gene inside E. coli cells, including the removal of the f-Met residue from the
nascent polypeptide. It is suggested that the pET system is a good tool
for the production of this protein, and the protein has no toxic effect
on the growth of E. coli.

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Fig. 2.
SDS-PAGE (4-15% gradient gel) of the
purified enzyme (lane 2). The following molecular mass
standards were used (lane 1): phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic
anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and
-lactalbumin (14.4 kDa).
|
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Unlike other AARE, the protein derived from P. horikoshii
needed a high concentration of NaCl to be dissolved. Therefore, the
purified protein solution used for the characterization contained 0.6 M NaCl. The molecular mass of the purified protein, as
determined by SDS-PAGE (Fig. 2), was consistent with that (72,390 Da)
calculated from the amino acid sequences. The molecular mass of the
protein determined by HPLC was about 150,000 Da (data not shown). The weighted-average molecular weight measured by light-scattering photometry using the dn/dc value determined for chicken gizzard myosin
was about 160,000. Therefore, the protein is likely a dimer structure,
instead of the four identical subunits found in mammals (9, 17). The
absorption coefficient (A280 nm) of the protein at 1% was determined to be 12.0.
Specificity of the Enzyme--
To examine the activity of this
protein, we used Ac-Leu-pNA, Ac-Ala-pNA,
Ac-Tyr-pNA, Leu-pNA, and Ala-pNA as
substrates. Table I shows the hydrolytic
activity (releasing of pNA) of the protein for them. At
85 °C and pH 5.4, the protein exhibited some hydrolytic activity for
Ac-Leu-pNA, Ac-Ala-pNA, Ac-Tyr-pNA and
no hydrolytic activity for Leu-pNA and Ala-pNA.
As shown in Table I, the protein also had hydrolytic activity for
acetylpeptides and formylpeptides. Analysis of the products by HPLC
revealed that the protein could only release the acylated amino acids
from acylpeptides. Therefore, this protein was concluded to be the AARE
from the thermophilic archaeon P. horikoshii. The
characteristics of this enzyme (hereafter referred to as AAREP) were
examined. The optimum pH of AAREP at 85 °C was between pH 4.8 and
5.5 (Fig. 3). The optimum temperature of
AAREP at pH 5.4 was about 90 °C (Fig.
4). Its specificity for small substrates
was different from those of AAREs in mammals (1, 17). Unlike the AARE
from rat, AAREP released Ac-Leu better than Ac-Ala from Ac-amino
acid-pNA; for most of the substrates used, the specific
activity of AAREP was higher than that of AARE from rat (Table I). The
activity decreased with increasing the residues of acylpeptides (Table
I). Table II shows that AAREP has similar
binding affinity for Ac-Leu-pNA, Ac-Ala-pNA,
Ac-Ala-Ala, and Ac-Ala-Ala-Ala-Ala. This result is different from that
of rat (17). The Km value obtained for Ac-Ala-Ala
was a little smaller than that for Ac-Ala-Ala-Ala-Ala (Table II). The
active site of AAREP seems to be suitable for relatively short acylpeptides. The hydrolytic activity of AAREP toward
-MSH was also examined under the above conditions. The NH2-terminal
amino acid sequence of
-MSH was not detected by the protein
sequencer after the incubation with AAREP. This result indicates that
AAREP cannot release Ac-Ser from
-MSH, unlike the AARE of
pig2 or rat (17). It is
peculated that AAREP is able to hydrolyze only short
acylpeptides.
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Table I
Substrate specificity of AAREP and HAAREP
The hydrolytic reaction was measured at 85 °C in 50 mM
sodium acetate buffer (pH 5.4) containing 0.6 M NaCl and
5% DMF. ND, activity was not detected.
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Fig. 3.
Effect of pH on the hydrolytic activity of
AAREP ( ) and HAAREP ( ) on Ac-Leu-pNA. The
hydrolytic activity was measured at 85 °C in 50 mM
sodium acetate buffer (pH 4-5.6), 50 mM sodium phosphate
buffer (pH 6-8), and 50 mM
NaH2PO4-Na2B4O7
buffer (pH 8-9), containing 0.6 M NaCl and 5% DMF. The
assay was measured for 10 min.
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Fig. 4.
Effect of the temperature on the hydrolytic
activity of AAREP ( ) and HAAREP ( ) on
Ac-Leu-pNA. The hydrolytic activity was measured in 50 mM sodium acetate buffer (pH 5.4) containing 0.6 M NaCl and 5% DMF. The assay was measured for 10 min.
Inset: Arrhenius plot.
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Table II
Kinetic parameters of AAREP and HAAREP
The hydrolytic reaction was measured at 85 °C in 50 mM
sodium acetate buffer (pH 5.4) containing 0.6 M NaCl and
5% DMF.
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Thermostability of the Enzyme--
Thermostability of the enzyme
was examined with CD and DSC. The CD spectrum in the far-UV region of
the enzyme was examined at 25 °C and 95 °C. The CD spectrum of
the enzyme at 95 °C was a little different from that at 25 °C
(Fig. 5). The intensity of the negative
ellipticity around 220 nm decreased slightly with increasing
temperature. The CD spectrum of AAREP at 95 °C was stable for
24 h.

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Fig. 5.
Far-UV CD spectra of AAREP at 25 °C ( )
and 95 °C ( ). The enzyme concentration was 0.23 mg/ml in 50 mM sodium acetate buffer (pH 5.4) containing 0.6 M NaCl. The path length of the quartz cell was 5.0 mm.
Three repetitions were completed to produce the data.
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Using DSC from 0 °C to 125 °C, we measured the heat capacity
changes of AAREP. We observed two peaks of heat capacity changes of
AAREP over 100 °C in the first scan (Fig.
6A) but no peak in the second
scan. Precipitate was observed after the first scan. The temperature of
the peaks was independent of the enzyme concentrations examined (0.1-2
mg/ml). This result indicates that the heat inactivation process of the
enzyme is irreversible and accompanied by aggregation. The two peaks
observed suggest that AAREP consists of two major domains as reported
by Miyagi et al. (12).

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Fig. 6.
DSC curves of AAREP (A) and
HAAREP (B) at protein concentrations of 1.06 and 0.90 mg/ml, respectively, in 50 mM sodium acetate buffer (pH
5.4) containing 0.6 M NaCl. The height of the small
blocks in the figure is 0.12 × 10 3 J/K. The
lower lines are base lines from buffer with no protein. Two
repetitions were completed to produce the data.
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These results indicate that incubating AAREP at 95 °C caused its
structure to begin unfolding, but the major conformation of the enzyme
remained stable from 0 °C to 100 °C.
Effect of Heating--
After incubating at 95 °C, we measured
the relative activity of AAREP at 85 °C to examine the effect of
heating. Incubating at 95 °C appeared to increase the relative
activity nearly 7-fold (Fig. 7). The
enzyme did not lose its increased activity upon cooling (4-25 °C),
suggesting that the activation was irreversible. This heat-activated
enzyme (hereafter referred to as HAAREP) was also stable at 95 °C
for 24 h.

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Fig. 7.
Activation of the enzyme by heat
treatment. AAREP (1.0 mg/ml) was incubated at 95 °C in 50 mM sodium acetate buffer (pH 5.4) containing 0.6 M NaCl. At the time shown, aliquots were taken out, and the
activities were measured in the same buffer at 85 °C using
Ac-Leu-pNA as substrate. Three repetitions were completed to
produce the data.
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From the light-scattering photometry, the molecular mass of HAAREP was
determined to be 260 kDa, and the spatial size of the associated
molecule was observed to be expanded in space compared with AAREP; its
z-average radius of gyration (RG) had
increased from less than 100 to more than 400. These molecular mass and RG values were virtually constant for nearly 5 days (250-264 kDa and RG = 416-445) at room
temperature, without significant decomposition of the molecule or
development of aggregation. The molecular mass value indicates that the
number of monomers constituting the associated molecule averages a
little more than 3 in this condition. However, the significant spacial
expansion (RG = 416-445) strongly suggests a
conformational change over the whole monomeric structural unit as a
result of heat treatment. By heating, the absorbance around 250-280 nm
was increased by 10 ± 1.3%, and the intensity of the negative
ellipticity of the CD around 220 nm was decreased slightly (Fig. 5).
The changes in the absorbance and CD were parallel to the change in the
activity of the enzyme. The NH2-terminal sequence of HAAREP
remained identical to that of AAREP. The rate of activation by heat
treatment was independent of the enzyme concentrations examined
(0.04-1.23 mg/ml). Therefore, it is deduced that the conformational
change by heat treatment alters the molecular character of monomer and
increases the activity. The NH2-terminal section of about
500 residues (12) in the enzyme might be related the conformational
change by heat treatment. We are continuing to investigate these
points.
In comparing the characteristics of the two enzymes, we found that
HAAREP had a higher optimum pH, above 7.0 (Fig. 3) and a higher optimum
temperature, 95 °C (Fig. 4). The relative specificity of HAAREP for
substrates was similar to that of AAREP, although HAAREP showed a
7-14-fold increase in specific activity (Table I). The activation
parameters of these enzymes were measured from 50-85 °C (Table
III). The temperature dependence on the
Km value of Ac-Leu-pNA for AAREP
(Km values at 60, 75, 80, 85, and 90 °C were
0.689 ± 0.21, 3.60 ± 0.66, 3.85 ± 0.91, 11.0 ± 6.3, and 18.0 ± 5.6 mM, respectively) was similar to that for HAAREP
(Km values at 60, 75, 80, 85, and 90 °C were
0.876 ± 0.24, 4.12 ± 0.51, 3.99 ± 0.66, 12.9 ± 9.0, and 19.7 ± 2.1 mM, respectively) (Table III). From the temperature
dependence on the kcat value of
Ac-Leu-pNA, the activation energy of HAAREP was found to be
greater than that of AAREP (Fig. 4 and Table III). Both
S
and
H
values of the activation were
increased by heat treatment. The shapes of the DSC curves of HAAREP
(Fig. 6B) and AAREP (Fig. 6A) were slightly
different from each other, but both enzymes seem to be stable below
100 °C. These results suggest that the conformational change in the
enzyme by heat treatment has an orienting effect on the catalytic
groups of the active site, making the enzyme more active at higher
temperatures. It is speculated that HAAREP is a stable intermediate
state between the native AAREP state and the heat-inactivated
(unfolded) state of the enzyme. Although we have no information about
the activity and structure of native AARE in P. horikoshii
cells, HAAREP is thought to be the dominant state of the enzyme in
P. horikoshii because of the organism's high optimum growth
temperature.
Until now, AARE has been found only in eukaryotic cells and thought to
be related to the initiation of protein synthesis (1, 21-23). The
existence of this enzyme in the archaeon P. horikoshii suggests that the initiation of protein synthesis in archaea is similar
to that in eukaryotic cells. From the fact that a number of eukaryotic
intracellular proteins are known to be
N
-acylated (24, 25), it is speculated
that many proteins of archaea are also
N
-acylated. Furthermore, the existence
of proteasomes in P. horikoshii (26, 27) suggests that the
action of the enzyme AAREP might be related to the
ubiquitin/ATP-dependent system of protein degradation (28-31). Archaea also contains aminoacylase (26, 32), which might play
an important role in the recycling of acylamino acids for protein
synthesis with the help of AARE.
In eukaryotic cells, a strong degree of genetic similarity between AARE
and aminoacylase was suggested by Jones et al. (33). In
P. horikoshii, however, the gene for aminoacylase was found at another locus in the genome (27), and AAREP did not share homology
or activity with aminoacylase.
AARE from mammals has been used to remove
N
-acylamino acid residues from
acylpeptides for protein sequencing at relatively low temperature
(37 °C). AAREP may not be used to remove
N
-acylamino acid residues of
relatively long acylpeptides. However, AAREP is expected to be used for
relatively short acylpeptides in sequence analysis at temperatures
higher than 90 °C.
Studies about the crystal structure, thermostability, and hydrolytic
mechanism of AAREP are in progress.
 |
ACKNOWLEDGEMENTS |
We thank T. Hashimoto, M. Jyutori, Dr. Y. Kosugi, Dr. S. Kawasaki, and Dr. S. Tsunasawa for great assistance in
the experiments in this study.
 |
FOOTNOTES |
*
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB009494.
§
To whom correspondence should be addressed. Fax: 81-298-54-6151;
E-mail: ishikawa{at}nibh.go.jp
1
The abbreviations used are: AARE, acylamino
acid-releasing enzyme; Ac-, N
-acetyl;
f-, N
-formyl; pNA,
p-nitroanilide;
-MSH,
-melanocyte-stimulating hormone;
PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid
chromatography; DMF, N, N-dimethylformamide; DSC,
differential scanning calorimetry; AAREP, AARE from P. horikoshii; HAAREP, heat-activated AAREP.
2
K. Ishikawa and S. Tsunasawa, unpublished
data.
 |
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