modulation of a chymotrypsin-type serine protease,Kentaro Shiraki1, Shigemi Norioka2, Shaoliang Li2, Kiyonobu Yokota3and Fumio Sakiyama2,* 1 School of Materials Science, Japan Advanced I
Trang 1modulation of a chymotrypsin-type serine protease,
Kentaro Shiraki1, Shigemi Norioka2, Shaoliang Li2, Kiyonobu Yokota3and Fumio Sakiyama2,*
1
School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan;2Institute 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–His210in 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 His210by 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 His210was responsible for isolation of the electrostatic interaction between His210and 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
Achromobacterprotease 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 4Murea
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
in close proximity to the active site The structural 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 His210and 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– His210prompted us to explore a possible contribution
of the p–p interaction to the enzymatic properties of API We have previously reported that the Trp169– His210pair 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– His210from 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: kshiraki@jaist.ac.jp
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)
Trang 2M A T E R I A L S A N D M E T H O D S
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 20mM Tris/HCl and 20mM Mes buffer
containing 0–1.5M NaCl to the desired final substrate
concentration After incubation for 10min at 37C, 2 mL
of the substrate solution was mixed with 100 lL of a 2-nM
enzyme solution The increase in fluorescence due to the
release of MCA was monitored at 440nm upon excitation
at 380 nm with a fluorescence spectrometer Hitachi F-4000
Values for the kinetic rate constant (kcat) and Michaelis
constant (Km) 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 lM
substrate in 20mM Tris/HCl and 20mM Mes buffers
containing 0–1.5MNaCl at 0.5 nMenzyme concentration
at 37C The increase in fluorescence of the released MCA
was monitored at 440nm upon excitation at 380nm 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 theINSIGHT II/DISCOVERsoftware The radius of the solvent probe was 1.4 A˚
Measurement of1H-NMR The pH-dependent1H-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% D2O and either 100 mM 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
1H-NMR spectra were measured on a JEOL Alpha 600 spectrometer equipped with a pulsed field gradient unit using the pulse sequence withWATERGATEsolvent suppres-sion To improve the signal-to-noise ratio, all spectra were recorded as an average of 16 000 scans
R E S U L T S
The pH-activity profiles of Trp169 and His210 mutants
In order to reveal the role of the Trp169–His210mutants in catalysis, Trp169 mutants replaced by Tyr (W169Y), Phe (W169F), His (W169H), Leu (W169L), Val (W169V), and Ala (W169A), His210mutants 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 (v0) was determined as a function of pH Fig 2 shows the v0vs pH profile of the API variants The enzymatic activity of chymotrypsin displays a bell-shaped
pH dependence; the acidic rim is at pK 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
His210in API is unique among
chymotrypsin-type serine proteases.
Trang 3alkaline rim is at pKa¼ 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 pK1 and pK2 and
their observed maximal rate constants, vmax1and vmax2
The pH-v0profile of wild-type API presented in Fig 2 fits
best at pK1¼ 6.0and pK2¼ 8.4 The pH-v0 profiles of
W169V and W169L showed a similar double-phase
sig-moidal distribution, while the acidic rim on the pH-v0
profiles shifted to neutral pH The pK2values of the Trp169
and His210variants were determined and are listed in
Table 1 pK2values of the Trp169 mutants lowered the
acidic rim by 0–0.9 pH units
mutants W169A–H210A and W169F–H210A showed profiles identical to that of the single mutant H210A The profile on the acidic rim of those His210variants is similar
to those of trypsin and chymotrypsin H210K had
pK¼ 8.6, while the mutants with uncharged residues at position 210 (H210A, H210S, W169F–H210A, and W169A–H210A) had pK¼ 6.3, indicating that His57 and His210should be tentatively assigned as the pKa6.0group and the pKa8.4 group, respectively
Energy minimization and pK2profile to determine the accessibility of the side-chain of His210
To understand the various pK2 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 pK2 showed a clear linear relationship (Fig 3B) The pK2 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 His210increased, the electrostatic interaction between His210and Asp113 weakened due to the increasing local dielectrostatic constant
1 H-NMR analysis in the region of low-barrier hydrogen bond
The pH-activity profiles were suggestive of a close relation-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 of1H-NMR (Fig 4)
A sharp proton signal was detected at around 16 p.p.m
at pH 9.1, which was assigned to 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 5.0and 4C merged into a single peak at 37C (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
H210A-W169F 3.8 ± 0.9 6.26c –
H210A-W169A 0.11 ± 0.07 6.31 c –
a
k cat /K m was determined using 20m M Tris/HCl buffer (pH 9.0).
b ASA of His210was obtained after simulation of structural
min-imization using INSIGHT II / DISCOVER cpK 2 values for
His210vari-ants were fitted to a single sigmoidal curve.
Trang 4The proton signal of His57 Ne2-Ser194 Oc was also
detected at around 14.0p.p.m at pH 9.1–6.9 The
His57 Ne2 proton signal disappeared below pH 6.0due
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
pK2 values remained constant at around pH 8.4 from 10mMNaCl to 1.3MNaCl (Fig 5B) These data indicate that the electrostatic interaction between Asp113 and His210was isolated from solvent
D I S C U S S I O N
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 His210is 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 20residues found in enzymes Due to its neutral pKa, 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 pKa¼ 6.5 in the pH-v0 curve However, the pH-v 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 His210for seven API variants at residue 169.
Fig 4 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 16.0p.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.
Trang 5titration curve (Fig 2) The pH-v0profile for wild-type API
appeared to be double phased, with the main curve at pH
8–10and a shoulder at pH 6–8, resulting from two ionizable
groups On the other hand, the pH-v0profiles of H210A and
H210S, which are chymotrypsin-type mutants, were clearly
different from that of wild-type API These results indicate
that His57 and His210may 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 His210converts the hydrogen
bonded network between Asp113 Od2 and His57 Nd1 into
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 pK2-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 104-fold [24] Therefore, the negatively charged Asp113 connected with the catalytic His57 Nd1 is 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, His210is 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 pKaof His210is 2 pH units higher than that of His57
is the buried charge interaction with Asp113 The shielding effect of Asp113-His210by 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 His210side-chain and it isolates His210from 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 His210side-chain This idea was confirmed by energy minimization (Fig 3) The charge interaction between Asp113 and His210is weakened with increasing solvent-accessibility of the His210side-chain In the protein interior, the dielectrostatic constant is lower than
on the protein surface, while the dielectrostatic constant in water is about 80and that in the protein interior is estimated
to be between 1 and 20[25] Accordingly, the pK2on 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 180m 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) and pK 2 vs NaCl concentration (s).
Trang 6side-chain at residue 210is 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, His210and
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 His210dominate, wild-type API
expresses the peptidase activity at a low level (state B)
However, full activity is not due to the electrostatic
interaction between His210and Asp113 Upon
deprotona-tion of His210with 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 pKaof
His210, is mainly determined by the type of side-chain at
residue 169 In the Trp169 mutants, His210deprotonates 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 chymo-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 His210to 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 His210interacts 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 pKaof His210, which is supported by the Trp169–His210stacking, suggesting that API has a catalytic quadruple apparatus, composed of Ser194, His57, Asp113 and His210, rather than a catalytic triad
A C K N O W L E D G E M E N T
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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