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: 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) 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. 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. 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(1987) The catalytic role of the active site aspartic acid in serine proteases. Science 237, 909–913. 25. Nakamura, H., Sakamoto, K. & Wada, A. (1988) A theoretical study of the dielectric constant of protein. Protein Eng. 2, 177–183. 4158 K. Shiraki et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Electrostatic role of aromatic ring stacking in the pH-sensitive modulation of a chymotrypsin-type serine protease, Achromobacter protease I Kentaro. Advanced Institute of Science and Technology, Ishikawa, Japan Achromobacter protease I (API) has a unique region of aromatic ring stacking with Trp169–His210 in