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

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

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

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

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

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

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

/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

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.

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

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).

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.
REFERENCES
1. Masaki, T., Nakamura, K., Isono, M. & Soejima, M. (1978) A
new proteolytic enzyme from Achromobacter lyticus M497-1.
Agric. Biol. Chem. 42, 1443–1445.
2. Masaki, T., Fujihashi, T., Nakamura, K. & Soejima, M. (1981)
Studies on a new proteolytic enzyme from Achromobacter lyticus
Fig. 6. Tentative charge state of His57, Asp113, and His210 in API. Wild-type API: state A, inactive state below pH 6.0; state B, low activity state
pH 6.0–8.6; state C, high activity state above pH 8.6. W169V: state A, inactive state below pH 6.0; state B, low activity state pH 6.0–7.8; state C,
high activity state above pH 7.8. H210S: state A, inactive state below pH 6.3; state B, active state above pH 6.3.
Ó FEBS 2002 Aromatic stacking in API (Eur. J. Biochem. 269) 4157
M497–1. II. Specificity and inhibition studies of Achromobacter.
Biochim. Biophys. Acta. 660, 51–55.
3. Masaki,T.,Tanabe,M.,Nakamura,K.&Soejima,M.(1981)
Studies on a new proteolytic enzyme from Achromobacter lyticus
M497–1. I. Purification and some enzymatic properties. Biochim.
Biophys. Acta. 660, 44–50.
4.Sakiyama,F.&Masaki,T.(1994)Lysylendopeptidaseof
Achromobacter lyticus. Methods Enzymol. 244, 126–137.
5. Ohara, T., Makino, K., Shinagawa, H., Nakata, A., Norioka, S. &
Sakiyama, F. (1989) Cloning, nucleotide sequence, and expression
of Achromobacter protease I gene. J. Biol. Chem. 264, 20625–

Nakamura, K.T. (1996) The crystal structure of ribonuclease
Rh from Rhizopus niveus at 2.0 A
˚
resolution. J. Mol. Biol. 255,
310–320.
16. Burley, S.K. & Petsko, G.A. (1985) Aromatic–aromatic interac-
tion: a mechanism of protein structure stabilization. Science 229,
23–28.
17. McGaughey, G.B., Gagne, M. & Rappe, A.K. (1998) p-Stacking
interactions. Alive and well in proteins. J. Biol. Chem. 273, 15458–
15463.
18. Burley, S.K. & Petsko, G.A. (1986) Amino–aromatic interactions
in proteins. FEBS Lett. 203, 139–143.
19. Chakrabarti, P. & Samanta, U. (1995) CH/, p interaction in the
packing of the adenine ring in protein structures. Advan. Protein
Chem. 39, 125–189.
20. Brandl, M., Weiss, M.S., Jabs, A.S., ¸ hnel,J.&Hilgenfeld,R.
(2001) C–H p-interaction in proteins. J. Mol. Biol. 307, 357–377.
21. Blow, D.M., Birktoft, J.J. & Hartley, B.S. (1969) Role of a buried
acidgroupinthemechanismofactionofchymotrypsin.Nature
221, 337–340.
22. Markley, J.L. & Westler, W.M. (1996) Protonation-state
dependency of hydrogen bond strengths and exchange rates in
a serine protease catalytic triad: bovine chymotrypsinogen
A. Biochemistry 35, 11092–11097.
23. Schechter, I. & Berger, A. (1967) On the size of the active site in
proteases. I. Papain. Biochem. Biopys. Res. Comm. 27, 157–162.
24. Craik, C.S., Roczniak, S., Largman, C. & Rutter, W.J. (1987) The
catalytic role of the active site aspartic acid in serine proteases.
Science 237, 909–913.