Tài liệu Báo cáo khoa học: The molecular surface of proteolytic enzymes has an important role in

The molecular surface of proteolytic enzymes has an important role

in stability of the enzymatic activity in extraordinary environments

Youhei Yamagata1

, Hiroshi Maeda1

, Tasuku Nakajima1 and Eiji Ichishima2

1

Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University,

Aoba-ku, Sendai, Japan; 2

Department of Biotechnology, Faculty of Engineering, Soka University, Hachioji, Tokyo, Japan

1It is scientifically and industrially important to clarify the

stabilizing mechanism of proteases in extraordinary envi￾ronments. We used subtilisins ALPI and Sendai as models

to study the mechanism. Subtilisin ALPI is extremely

sensitive to highly alkaline conditions, even though the

enzyme is produced by alkalophilic Bacillus, whereas sub￾tilisin Sendai from alkalophilic Bacillus is stable under

conditions of high alkalinity. We constructed mutant

subtilisin ALPI enzymes by mutating the amino acid

residues specific for subtilisin ALPI to the residues at the

corresponding positions of amino acid sequence alignment

of alkaline subtilisin Sendai. We observed that the two

mutations in the C-terminal region were most effective for

improving stability against surfactants and heat as well as

high alkalinity. We predicted that the mutated residues are

located on the surface of the enzyme structures and, on

the basis of three-dimensional modelling, that they are

involved in stabilizing the conformation of the C-terminal

region. As proteolytic enzymes frequently become inactive

due to autocatalysis, stability of these enzymes in an

extraordinary environment would depend on the confor￾mational stability of the molecular surface concealing

scissile peptide bonds. It appeared that the stabilization of

the molecular surface structure was effective to improve the

stability of the proteolytic enzymes.

Keywords: alkalophilic alkaline resistance; Bacillus; mole￾cular surface structure; serine protease; subtilisin.

There have been several studies of the difference aspects

of proteolytic enzymes and they have been used in various

industrial fields. In particular, subtilisins, serine proteases

from a variety of Bacillus species, are some of the most

investigated enzymes [1,2]. Subtilisins are classified into

three groups, the neutral subtilisins, the alkaline subtilisins

and
the ALPI-type subtilisin (Fig. 1) [3]. The neutral

subtilisins consist of the subtilisins from neutrophilic

Bacillus such as subtilisin BPN¢ [4], Carlsberg [5], E [6],

and NAT [7]. The alkaline subtilisin group contains the

enzymes from alkalophilic Bacillus such as subtilisin YaB

[8], no. 221 protease [9], Savinase [10], subtilisin Sendai

(Sendai) [11]. Subtilisin ALPI (ALPI) from alkalophilic

Bacillus NKS-21 [3] is only member of the ALPI-type

subtilisins. ALPI is extremely sensitive to high alkaline

conditions, even though the enzyme is produced by an

alkalophilic Bacillus. On the other hand, Sendai from

alkalophilic Bacillus sp. G-825-6, categorized as an

alkaline subtilisin, is very stable under highly alkaline

conditions.

Maeda et al. reported that the inactivation of subtilisin

ALPI at high alkalinity was caused by the instability of

its molecular surface structure and autolysis in the

N-terminal region and/or the C-terminal region [12,13].

We hypothesized that the divergence of the properties of

ALPI from the alkaline subtilisins might depend on the

structure of the enzyme. In particular, the instability of

ALPI in highly alkaline conditions might be caused by

the existence of consensus amino acid sequences of

ALPI and the neutral subtilisins and/or the peculiar

residues in the amino acid sequence of ALPI. We

selected 12 consensus amino acid residues from the

amino acid sequence alignment of ALPI and the neutral

subtilisins. These candidate residues did not occur at the

corresponding positions of the alkaline subtilisins. Fur￾thermore, on the basis of the predicted three-dimensional

structure of ALPI, we believed that the C-terminal

region was located on the molecular surface and was

exposed to the solvent phase; therefore two unique

residues in the C-terminal region were replaced by the

residues at corresponding positions of amino acid

sequence of Sendai. As a result of analysing the mutant

ALPI s, two amino acid residues in the C-terminal

region were found to play important roles in maintaining

stability in highly alkaline conditions. The double muta￾tions prolonged the half-lifetime by more than 120-fold.

The substitutions of the amino acid residues also

improved the stability of the enzyme to detergents and

heat.

Correspondence to: Y. Yamagata, Laboratory of Molecular

Enzymology, Division of Life Science, Graduate School of

Agricultural Science, Tohoku University, 1-1, Tsutsumidori￾Amamiyamachi, Aoba-ku, Sendai, Japan, 981-8555.

Fax: + 81 22717 8778, Tel.: + 81 22717 8776,

E-mail: [email protected]

Abbreviations: ALPI, subtilisin ALPI; Sendai, subtilisin Sendai;

Suc-Ala-Ala-Pro-Phe-MCA, succinyl-L-alanyl-L-alanyl-L-proryl-L￾phenylalanyl-4-methylcoumaryl-7-amide; DSC, differential scanning

calorimetry; LAS, sodium lauryl benzene sulfate.

Enzymes: Subtilisin ALPI (EC 3.4.21.64); subtilisin Sendai

(EC 3.4.21.64).

(Received 8 May 2002, revised 23 July 2002,

accepted 26 July 2002)

Eur. J. Biochem. 269, 4577–4585 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03153.x