Tài liệu Báo cáo khoa học: Electrostatic role of aromatic ring stacking in the pH-sensitive

Electrostatic role of aromatic ring stacking in the pH-sensitive

modulation of a chymotrypsin-type serine protease,

Achromobacter protease I

Kentaro Shiraki1

, Shigemi Norioka2

, Shaoliang Li2

, Kiyonobu Yokota3 and Fumio Sakiyama2,*

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–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.

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

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: [email protected]

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)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03110.x