Tài liệu Báo cáo khoa học: Electrostatic role of aromatic ring stacking in the pH-sensitive modulation of a chymotrypsin-type serine protease, Achromobacter protease I pdf

Electrostatic role of aromatic ring stacking in the pH-sensitive
modulation of a chymotrypsin-type serine protease,
Achromobacter
protease I
Kentaro Shiraki
1
, Shigemi Norioka
2
, Shaoliang Li
2
, Kiyonobu Yokota
3
and Fumio Sakiyama
2,
*
1
School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan;
2
Institute for Protein Research,
Osaka University, Suita, Osaka, Japan;
3
School of Knowledge Science, Japan Advanced Institute of Science and Technology,
Ishikawa, Japan
Achromobacter protease I (API) has a unique region of
aromatic ring stacking with Trp169–His210 in close proxi-
mity to the catalytic triad. This paper reveals the electrostatic
role of aromatic stacking in the shift in optimum pH to the
alkaline region, which is the highest pH range (8.5–10)
among chymotrypsin-type serine proteases. The pH-activity
profile of API showed a sigmoidal distribution that appears
at pH 8–10, with a shoulder at pH 6–8. Variants with

smaller amino acid residues substituted for Trp169 had
lower pH optima on the acidic side by 0–0.9 units. On the
other hand, replacement of His210 by Ala or Ser lowered the
acidic rim by 1.9 pH units, which is essentially identical to
that of chymotrypsin and trypsin. Energy minimization for
the mutant structures suggested that the side-chain of
Trp169 stacked with His210 was responsible for isolation of
the electrostatic interaction between His210 and the catalytic
Asp113 from solvent. The aromatic stacking regulates the
low activity at neutral pH and the high activity at alkaline
pH due to the interference of the hydrogen bonded network
in the catalytic triad residues.
Keywords: aromatic stacking; catalytic triad; pH-depend-
ence; serine protease.
Achromobacter protease I (API; EC 3.4.21.50) is a chymo-
trypsin-type serine protease that Achromobacter lyticus
M497-1 secretes extracellularly [1]. We have studied the
structure–function relationship of API because of its
attractive properties: (a) restricted lysyl-bond specificity,
including the Pro–Lys bond; (b) one order of magnitude
higher activity than bovine trypsin; (c) broad optimum pH
range in the alkaline region (pH 8.5–10.5); and (d) high
stability against denaturing conditions, including 4
M
urea
and 0.1% SDS [2–4].
API is synthesized as a 658-residue preprotein that is
autocatalytically activated [5,6]. Mature API is a 268-
residue monomer [7]. The amino acid sequence identity
between API and bovine trypsin is as low as 20%.

However, X-ray crystallographic analysis of API at 1.2 A
˚
resolution (protein data bank code 1arb) revealed that
the apparent secondary structure of the protein is quite
similar to that of chymotrypsin-type serine proteases
(Fig. 1). The catalytic triad residues Asp113, His57, and
Ser194 in API are placed at an identical location to those
of chymotrypsin and bovine trypsin. The catalytic triad
residues and the substrate binding S1 pocket are located
incloseproximitytotheactivesite.Thestructural
alignment of the catalytic triad residues and substrate
binding S1 pocket in API is not special but quite typical.
The noticeable difference is a region of aromatic stacking
between Trp169 and His210 (Fig. 1). The two aromatic
planes stack at a distance of 3.5 A
˚
, and the shortest
distance between the imidazole ring of His210 and the
atoms of Asp113 is 3.2 A
˚
. The substrate binding subsite
in API is composed of His210-Gly211-Gly212, while that
in chymotrypsin-type serine proteases is widely conserved,
and consists of Ser–Trp–Gly [8,9]. The detection of the
unique structural arrangement mediated by Trp169–
His210 prompted us to explore a possible contribution
of the p–p interaction to the enzymatic properties of
API. We have previously reported that the Trp169–
His210 pair functions in the high catalytic activity of this
protease at pH9 [10]. Further interest in the aromatic

stacking is in the role of the electrostatic properties in
enzymatic catalysis of API, and in distinguishing the
functionally catalytic quadruple Ser194–His57–Asp113–
His210 from the usual catalytic triad Ser194–His57–
Asp113.
In this paper, we report the contribution of the electro-
static interaction of Asp113–His210, which is supported by
Trp169, in the pH-sensitive modulation of activity as
unravelled by analysis of the kinetics of single and double
mutants with substitutions at positions 169 and 210. This
result implies a novel function for p–p stacking in the
reactive site of this enzyme.
Correspondence to K. Shiraki, School of Materials Science,
Japan Advanced Institute of Science and Technology, 1-1 Asahidai,
Tatsunokuchi, Ishikawa, 923-1292, Japan.
E-mail:
Abbreviations:API,Achromobacter protease I; ASA, accessible surface
area; Boc, t-butoxycarbonyl; MCA, 4-methylcoumaryl-7-amide;
VLK-MCA, Boc-Val-Leu-Lys-MCA.
Enzyme: Achromobacter protease I (EC 3.4.21.50).
*Present address: International Buddhist University, 3-2-1
Gakuenmae, Habikino, Osaka 583–8501, Japan.
(Received 14 March 2002, revised 8 July 2002,
accepted 11 July 2002)
Eur. J. Biochem. 269, 4152–4158 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03110.x
MATERIALS AND METHODS
Materials
The substrate peptide t-butoxycarbonyl-Val-Leu-Lys4-
methylcoumaryl-7-amide (VLK-MCA) was purchased
from Peptide Institute Inc. (Osaka, Japan). All restriction

and modification enzymes were from TAKARA Co. Ltd.
(Kyoto, Japan). All other chemicals were from commercial
suppliers and were of the highest analytical grade.
Single-stranded DNA for mutagenesis was obtained from
plasmid pKYN200 [5]. The mutagenesis was performed
according to the Uracil-DNA mediated method [11]. The
mutant genes encoding W169Y, W169F, W169L, W169V,
W169A, H210S, H210A, and H210K were constructed as
described previously [10]. The double mutant genes enco-
ding W169A-H210A and W169F-H210A were constructed
from single mutant genes using appropriate restriction
enzymes and ligase. Transformants of Escherichia coli strain
JA221 cells were grown on Luria–Bertani medium supple-
mented with 50 lgÆmL
)1
ampicillin. The expression and
purification of wild-type and mutants was carried out as
described previously [6]. The amount of purified protein was
 0.5–0.8 mg from 2-L cultures.
Determination of kinetic parameters
The substrate solution in 1% dimethylformamide was
diluted with 20 m
M
Tris/HCl and 20 m
M
Mes buffer
containing 0–1.5
M
NaCl to the desired final substrate
concentration. After incubation for 10 min at 37 °C, 2 mL

of the substrate solution was mixed with 100 lLofa2-n
M
enzyme solution. The increase in fluorescence due to the
release of MCA was monitored at 440 nm upon excitation
at 380 nm with a fluorescence spectrometer Hitachi F-4000.
Values for the kinetic rate constant (k
cat
) and Michaelis
constant (K
m
) were obtained from the initial velocity
on theoretical curves calculated by nonlinear regression
analysis.
pH-activity profiles for API mutants were determined as
follows. Assay buffers and other conditions were: 100 l
M
substrate in 20 m
M
Tris/HCl and 20 m
M
Mes buffers
containing 0–1.5
M
NaCl at 0.5 n
M
enzyme concentration
at 37 °C. The increase in fluorescence of the released MCA
was monitored at 440 nm upon excitation at 380 nm and
the value of initial velocity was determined.
Energy minimization for Trp169 mutants

To determine the structure of Trp169 mutants, an energy
minimization program was utilized based on the X-ray
crystal structure of wild-type API. The coordinates for the
API variants were taken from PDB file code 1arb. The
appropriate residues were changed at the site of the mutation
and all hydrogens were explicitly treated in the protein
models. The computer program
INSIGHT II/DISCOVER
(Accelrys Inc., San Diego, CA, USA) was used for energy
minimization. The solvent accessible surface areas (ASA) of
individual residues in the API variants were calculated with
the
INSIGHT II/DISCOVER
software. The radius of the solvent
probe was 1.4 A
˚
.
Measurement of
1
H-NMR
The pH-dependent
1
H-NMR of wild-type API was meas-
ured in order to measure the hydrogen bonds between the
catalytic residues. Sample solutions containing 5 mgÆmL
)1
protein in 10% D
2
O and either 100 m
M

Tris/HCl
(> pH 6.9) or Mes (< pH 6.8) were prepared. The
0.5-mL samples were held in 5 mm diameter NMR tubes.
1
H-NMR spectra were measured on a JEOL Alpha 600
spectrometer equipped with a pulsed field gradient unit
using the pulse sequence with
WATERGATE
solvent suppres-
sion. To improve the signal-to-noise ratio, all spectra were
recorded as an average of 16 000 scans.
RESULTS
The pH-activity profiles of Trp169 and His210 mutants
In order to reveal the role of the Trp169–His210 mutants in
catalysis, Trp169 mutants replaced by Tyr (W169Y), Phe
(W169F), His (W169H), Leu (W169L), Val (W169V), and
Ala (W169A), His210 mutants replaced by Ala (H210A),
Ser (H210S), and Lys (H210K), and double mutants
W169F–H210A and W169A–H210A were constructed.
Peptidase activity was determined using VLK-MCA as
the substrate and the increase in fluorescence of the released
MCA was monitored. The maximum peptidase activity at
each respective pH (v
0
) was determined as a function of pH.
Fig. 2 shows the v
0
vs. pH profile of the API variants. The
enzymatic activity of chymotrypsin displays a bell-shaped
pH dependence; the acidic rim is at pK

a
¼ 6.5 and the
Fig. 1. Stick models of the reactive site in
bovine trypsin and API. The catalytic triad
residues of trypsin and API are Ser195–His57–
Asp102 and Ser194–His57–Asp113, respect-
ively. The substrate-binding subsite residues of
trypsin and API are Ser214–Trp215–Gly216
and His210–Gly211–Gly212, respectively. S1
pocket is the substrate binding site for the side-
chain of Lys (API) or Lys and Arg (trypsin).
The aromatic stacking between Trp169 and
His210 in API is unique among chymotrypsin-
type serine proteases.
Ó FEBS 2002 Aromatic stacking in API (Eur. J. Biochem. 269) 4153
alkaline rim is at pK
a
¼ 8.8. On the other hand, the activity
of API did not decrease above pH 10.0. Wild-type API
shows low activity at pH 6–8 and high activity at pH 8–10.
The double-phase curve was well fitted to the equation that
includes two ionizable groups bearing pK
1
and pK
2
and
their observed maximal rate constants, v
max1
and v
max2

.
The pH-v
0
profile of wild-type API presented in Fig. 2 fits
best at pK
1
¼ 6.0 and pK
2
¼ 8.4. The pH-v
0
profiles of
W169V and W169L showed a similar double-phase sig-
moidal distribution, while the acidic rim on the pH-v
0
profiles shifted to neutral pH. The pK
2
values of the Trp169
and His210 variants were determined and are listed in
Table 1. pK
2
values of the Trp169 mutants lowered the
acidic rim by 0–0.9 pH units.
In contrast, when His210 was replaced by Ala (H210A),
the optimum pH shifted dramatically toward the neutral
region (Fig. 2). The single mutant H210S and the double
mutants W169A–H210A and W169F–H210A showed
profiles identical to that of the single mutant H210A. The
profile on the acidic rim of those His210 variants is similar
to those of trypsin and chymotrypsin. H210K had
pK ¼ 8.6, while the mutants with uncharged residues at

position 210 (H210A, H210S, W169F–H210A, and
W169A–H210A) had pK ¼ 6.3, indicating that His57 and
His210 should be tentatively assigned as the pK
a
6.0 group
and the pK
a
8.4 group, respectively.
Energy minimization and p
K
2
profile to determine
the accessibility of the side-chain of His210
To understand the various pK
2
values of the Trp169
variants, an energy minimization calculation was performed
using
INSIGHT II
/
DISCOVER
. For W169Y, W169F, and
W169H mutants, the side-chain at position 169 remained
parallel with the side-chain of His210. On the other hand, a
small side-chain at position 169, typically W169V and
W169A, deviates from the original position. In the struc-
tural deviation, the solvent ASA of the side-chain of His210
increased with the decrease in size of the side-chain at
residue 169 (Table 1 and Fig. 3A). However, the ASAs of
Asp113 and His57 remained constant when the side-chain at

residue 169 was changed (Fig. 3A). These results suggest
that the side-chain at residue 169 is responsible for the
solvent accessibility of His210.
The size of the side-chain at residue 169 and the pK
2
showed a clear linear relationship (Fig. 3B). The pK
2
increased as the size of the residue at position 169 increased.
The effect of the size of the residue at position 169 may be
related to the solvent accessibility of the side-chain of
His210 (Fig. 3C). When the accessibility of the side-chain of
His210 increased, the electrostatic interaction between
His210 and Asp113 weakened due to the increasing local
dielectrostatic constant.
1
H-NMR analysis in the region of low-barrier hydrogen
bond
ThepH-activityprofilesweresuggestiveofacloserelation-
ship of the ionization states in both His57 and His210. To
explore this possibility,we attempted to titrate the His57 Nd1
and Ne2 protons by means of
1
H-NMR (Fig. 4).
A sharp proton signal was detected at around 16 p.p.m.
at pH 9.1,which was assignedto the His57 Nd1-Asp113 Od2
proton based on the fact that the proton resonance of
His57 Nd1 in the catalytic triad is usually shifted approxi-
mately 5 p.p.m. down-field from the normal histidine NH
proton [12,13]. The single proton signal appeared at 15.8–
16.1 p.p.m. at pH > 8.2 and split into two signals at 16.4

and 15.8 p.p.m. at pH 8.2–5.0. With increasing temper-
ature, the two split proton signals at pH 5.0 and 4 °C
merged into a single peak at 37 °C (Fig. 4). The data
indicate that the split signals were originated by the one
proton, His57 Nd1-Asp113 Oc2, i.e. at high temperature,
the interchange rate of the proton between His57 Nd1-
Asp113 Oc2 may be too fast to monitor as the split signals,
while at low temperature, that of the interchange rate is too
slow to monitor as the single one.
Fig. 2. The relative pH-activity profiles of wild-type API (d), W169L
(s), W169V (m), H210A (n), H210S (.), and H210A-W169A (,)
with 180 m
M
NaCl.
Table 1. Kinetic parameters of API variants as obtained with Boc-Val-
Leu-Lys-MCA as substrate monitored at 37 °C.
Enzyme k
cat
/K
m
(l
M
)1
Æs
)1
)
a
pK
2
ASA of

His210 (A
˚
2
)
b
Wild-type 44 ± 9 8.41 41.1
W169Y 19 ± 3 8.30 43.1
W169F 20 ± 5 8.39 45.3
W169L 11 ± 3 8.04 44.6
W169H 4.0 ± 0.7 7.75 48.2
W169V 2.8 ± 1.1 7.83 57.3
W169A 0.23 ± 0.07 7.51 70.3
H210A 35 ± 9 6.32
c
–
H210S 74 ± 18 6.32
c
–
H210A-W169F 3.8 ± 0.9 6.26
c
–
H210A-W169A 0.11 ± 0.07 6.31
c
–
H210K 0.01 ± 0.02 8.58
c
–
a
k
cat

/K
m
was determined using 20 m
M
Tris/HCl buffer (pH 9.0).
b
ASA of His210 was obtained after simulation of structural min-
imization using
INSIGHT II
/
DISCOVER
.
c
pK
2
values for His210 vari-
ants were fitted to a single sigmoidal curve.
4154 K. Shiraki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The proton signal of His57 Ne2-Ser194 Oc was also
detected at around 14.0 p.p.m. at pH 9.1–6.9. The
His57 Ne2 proton signal disappeared below pH 6.0 due
to the protonation of His57 Ne2. These results do not
contradict the pH-activity profile of API shown in Fig. 2.
Ion strength dependence of the pH-activity curve of API
The pH-activity profiles depending on NaCl concentration
were determined as part of the investigation into the
shielding from solvent of the electrostatic interaction
between Asp113 and His210. With increasing NaCl, the
maximum activity decreased, while the shape of the pH-
activity profile was not changed essentially (Fig. 5A). The

pK
2
values remained constant at around pH 8.4 from
10 m
M
NaCl to 1.3
M
NaCl (Fig. 5B). These data indicate
that the electrostatic interaction between Asp113 and
His210 was isolated from solvent.
DISCUSSION
Aromatic ring stacking in the active sites of enzymes has
been reported and possible connections with their catalytic
functions have been considered [14,15]. In most cases,
however, aromatic stacking is formed in a perpendicular
orientation [16,17]. The parallel orientation of the imida-
zole–indole pair formed between Trp169 and His210 is the
first case found in the active sites of serine proteases.
Although the role of a proton donor for the imidazole and
indole side-chains has been suggested from database
analyses [16,18–20], we have been interested in this unique
aromatic stacking as a possible molecular mechanism in
enzyme catalysis.
Electrostatic interaction between Asp113 and His210
Histidine is one of the most functional amino acids among
the 20 residues found in enzymes. Due to its neutral pK
a
,
histidine often plays an important function as a hydrogen
bond donor and acceptor, and as the positively charged

member of a salt bridge. For serine proteases, His57 is also a
key residue in proteolytic catalysis [21]. The enzymatic
activity of chymotrypsin displays a typical titration curve;
the protonated-deprotonated equilibrium of His57 is
responsible for pK
a
¼ 6.5 in the pH-v
0
curve. However,
the pH-v
0
profile of wild-type API did not fit the typical
Fig. 3. The relationship between pK
2
and ASA. (A) Solvent accessible surface area of His57 (d), Asp113 (s), and His210 (j) for seven API variants
at residue 169. (B) pK
2
vs. volume at 169 residues for seven API variants at residue 169. (C) pK
2
vs. solvent accessible surface areas of His210 for
seven API variants at residue 169.
Fig. 4. pH- and temperature-dependent NMR. Left and Middle: pH-
dependent
1
H-NMR spectra of wild-type API at 4 °C. A dotted line is
placed at 16.0 p.p.m. Peaks A and B represent the tentative
His57 Nd1-Asp113 Oc proton. Peak C represents the tentative
His57 Ne2-Ser194 Oc proton. Right: temperature-dependent
1
H-NMR spectra of the wild-type API at pH 5.0.

Ó FEBS 2002 Aromatic stacking in API (Eur. J. Biochem. 269) 4155
titration curve (Fig. 2). The pH-v
0
profile for wild-type API
appeared to be double phased, with the main curve at pH
8–10 and a shoulder at pH 6–8, resulting from two ionizable
groups. On the other hand, the pH-v
0
profiles of H210A and
H210S, which are chymotrypsin-type mutants, were clearly
different from that of wild-type API. These results indicate
that His57 and His210 may be assigned as the two ionizable
groups related to the catalytic activity. These results
prompted us to propose a new catalytic mechanism as
follows.
The hydrogen-bonded network in the catalytic triad in
serine proteases is a well-known catalytic apparatus [21,22].
First, deprotonated His57 Ne2 is responsible for the
expression of activity [23]. Next, the buried hydrogen bond
between His57 Nd1 and Asp113 Od2 is constructed and it
enhances the basicity at His57 Ne2. His57 Ne2 enhances
the nucleophilicity of the Ser194 hydroxyl oxygen. Accord-
ingly, the Asp113–His57 diad is primarily important for the
expression of the nucleophilicity of the catalytic Ser194. The
side-chain of Asp113 is located 3.2 A
˚
from the side-chain of
His210. If His210 maintains its protonated form, the
Asp113 Od2–His57 Nd1 interaction is weakened by the
electrostatic interaction between Asp113 and His210. With

increasing pH, deprotonated His210 converts the hydrogen
bonded network between Asp113 Od2andHis57Nd1into
the normal strong form, the nucleophilicity of Ser194 Oc is
increased, and the activity of API is expressed.
Trp169 isolates Asp113–His210 electrostatic interaction
from solvent
The plot of the pK
2
-ASA of His210 (Fig. 3C) is considered as
follows. The role and importance of the aspartate in the
catalytic triad is not fully understood because several serine
proteases do not have an aspartate as the catalytic apparatus.
However, for chymotrypsin-type serine proteases, the
replacement of this aspartate with an alanine diminishes
protease activity 10
4
-fold [24]. Therefore, the negatively
charged Asp113 connected with the catalytic His57 Nd1is
necessary for the functional form of the catalytic triad. In a
majority of other serine proteases, Asp113 (Asp102 for
trypsin number) forms a solvent-inaccessible hydrogen bond
with the side-chain of a conserved serine at the position of
subsite S1. In API, His210 is also located in a solvent-
inaccessible position and interacts with the negatively
charged Asp113 at distance of 3.2 A
˚
. One of the reasons
that the pK
a
of His210 is 2 pH units higher than that of His57

is the buried charge interaction with Asp113. The shielding
effect of Asp113-His210 by Trp169 was supported by the
independency of the ionic strength of the pH-activity curve
(Fig. 5).
In the X-ray crystal structure, the Trp169 side-chain is
located on the outside of the His210 side-chain and it isolates
His210 from solvent. The solvent ASA of the side-chain of
Trp169 is 127 A
˚
2
, which is much greater than that of all other
residues in API, in addition to His210 (41 A
˚
2
) and Asp113
(2 A
˚
2
). Therefore, replacing Trp169 by other small residue
increases the solvent ASA of the His210 side-chain. This idea
was confirmed by energy minimization (Fig. 3). The charge
interaction between Asp113 and His210 is weakened with
increasing solvent-accessibility of the His210 side-chain. In
the protein interior, the dielectrostatic constant is lower than
on the protein surface, while the dielectrostatic constant in
water is about 80 and that in the protein interior is estimated
to be between 1 and 20 [25]. Accordingly, the pK
2
on the
acidic rim of the Trp169 mutants decreased with decreasing

size of the residue at 169 (Fig. 3).
Although the structural arrangement of this stacking
implies that the interaction between the imidazole and the
electron-rich indole ring is essentially electrostatic, the
Fig. 5. Ionic strength dependent of the pH-activity curve of API. (A) Titration curves with 180 m
M
NaCl (d), 500 m
M
NaCl (h), and 1.0
M
NaCl
(n). (B) Relative activity with various concentrations of NaCl at pH 9.0 (d)andpK
2
vs. NaCl concentration (s).
4156 K. Shiraki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
side-chain at residue 210 is dispensable, as shown by the fact
that H210A and H210S are as active as native API with
VLK-MCA as a substrate (Table 1). This means that
Trp169 does not play a role as an electron-rich entity but as
a large planar hydrophobic entity that can effectively shield
the side-chain of residue 210.
Molecular mechanism of aromatic stacking
for the optimum pH shift
Fig. 6 shows the charged state of key residues involved in
the catalytic activity of API. For wild-type API, His210 and
His57 are protonated at pH < 6.0 (state A). State A
represents inactive API due to the presence of positive
charges on His57. At pH 6.0–8.6, where unprotonated
His57 and protonated His210 dominate, wild-type API
expresses the peptidase activity at a low level (state B).

However, full activity is not due to the electrostatic
interaction between His210 and Asp113. Upon deprotona-
tion of His210 with increasing pH, the suppressed activity is
released and the protease exhibits a six- to sevenfold higher
activity than that at neutral pH (state C). The structural
change from state B to state C, which relates to the pK
a
of
His210, is mainly determined by the type of side-chain at
residue 169. In the Trp169 mutants, His210 deprotonates at
a lower pH compared to that for wild-type API, due to the
increased solvent accessibility of the electrostatic interaction
Asp113–His210. For example, His210 in the W169V variant
deprotonates at pH 7.8 and expresses full activity as the
respective mutant. On the other hand, the pH-activity
profile of H210S is determined only by His57. The
molecular mechanisms of the pH dependent activities of
the H210A and H210S mutants are identical to chymo-
trypsin and trypsin, i.e. the activity is expressed by removing
the His57 Ne2 proton.
A unique histidine at subsite S1 that performs a
protonation–deprotonation control device is also a novel
mechanism among serine proteases. The close position of
His210 to Asp113 guides us to a new way of thinking about
the functional role of the former ionizable aromatic amino
acid. The pH optimum mechanism in API results from two
things: (a) positively charged His210 interacts with nega-
tively charged Asp113; and (b) Trp169 isolates the electro-
static interaction from solvent. The pH optimum shift in the
alkaline region results from the high pK

a
of His210, which is
supported by the Trp169–His210 stacking, suggesting that
API has a catalytic quadruple apparatus, composed of
Ser194, His57, Asp113 and His210, rather than a catalytic
triad.
ACKNOWLEDGEMENT
We are grateful to Dr. T. Yamazaki for NMR measurements, Y. Yagi
for the amino acid analysis, and Y. Yoshimura for the sequence analysis.
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