Substrate-dependent activation of thermolysin by salt

Article abstract of DOI:10.1271/bbb.68.1811

Salt-activation of thermolysin was examined using a positively charged fluorescent substrate, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu- [N³-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH ₂ [MOCAc-PLGL(Dpa)AR]. Thermolysin activity increased in a biphasic exponential fashion and was 40 times higher in the presence of 4 M NaCl than in its absence. The degree of activation at X M NaCl was expressed as 4.7 ^x when [NaCl]₀ < 0.5 M and 2.3^x when [NaCl]₀ > 0.5 M respectively.

Full text of DOI:10.1271/bbb.68.1811

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Substrate-dependent Activation of Thermolysin by
Salt
a
a
a
Hiroshi ONEDA , Yuko MUTA & Kuniyo INOUYE
a
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto
University
Published online: 22 May 2014.
To cite this article: Hiroshi ONEDA, Yuko MUTA & Kuniyo INOUYE (2004) Substrate-dependent Activation of Thermolysin by
Salt, Bioscience, Biotechnology, and Biochemistry, 68:8, 1811-1813, DOI: 10.1271/bbb.68.1811
To link to this article: http://dx.doi.org/10.1271/bbb.68.1811
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Biosci. Biotechnol. Biochem., 68 (8), 1811–1813, 2004
Note
Substrate-dependent Activation of Thermolysin by Salt
y
Hiroshi ONEDA, Yuko MUTA, and Kuniyo INOUYE
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
Received April 26, 2004; Accepted June 1, 2004
Salt-activation of thermolysin was examined using a
positively charged fluorescent substrate, (7-methoxy-
reaction rate (v) was expressed as kcat [E] [S]=Km, and
o
ꢀ
1
ꢀ1
thermolysin showed k =K value of 75.0 mM
s at
cat
m
3
ꢁ
coumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N -(2,4-
pH 7.5, 25 C.
dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-
NH2 [MOCAc-PLGL(Dpa)AR]. Thermolysin activity
increased in a biphasic exponential fashion and was 40
times higher in the presence of 4 M NaCl than in its
absence. The degree of activation at x M NaCl was
Figure 1 shows activation of thermolysin by NaCl in
the hydrolysis of MOCAc-PLGL(Dpa)AR. Thermolysin
activity (v) increased in a biphasic exponential fashion
with increases in NaCl concentration, as is clearly
shown by the log v vs. [NaCl] plots. The v values at 0 M
o
x
x
ꢀ1
expressed as 4:7 when [NaCl] < 0:5 M and 2:3 when
o
and 4 M NaCl were 0:40 ꢂ 0:02 and 15:9 ꢂ 0:9 nM s
[NaCl] > 0:5 M respectively.
o
respectively. Activation behavior was analyzed by the
4
)
method introduced previously. The degree of activa-
tion in the presence of x M NaCl is expressed as v =v ,
Key words: metalloproteinase; thermolysin; salt-activa-
tion; halophilicity
x
o
where vx and vo are reaction rates at x M and 0 M NaCl,
x
x
determined to be 4:7 at x < 0:5 and 2:3 at x > 0:5
respectively. The parameters of salt-activation of ther-
molysin obtained with FAGLA and ZDFM, and that of
human matrix metalloproteinase 7 (MMP-7), a collage-
nase closely related to thermolysin, are summarized in
Table 1. Salt-activation of the hydrolysis of a neutral
substrate, FAGLA, is characterized by a single expo-
nential curve, whereas that of a negatively charged
substrate, ZDFM, is characterized by a biphasic expo-
nential one. It should be noted that the vx=vo value at
x < 0:5 obtained with MOCAc-PLGL(Dpa)AR is higher
and the value with ZDFM lower than those at x > 0:5,
probably due to the repulsive and attractive electrostatic
interactions between thermolysin and the substrates
respectively. It is suggested that thermolysin prefers
negatively charged substrates at low concentrations of
salts. The vx=vo value with FAGLA is almost the same
as that with ZDFM at x > 0:5, suggesting that salt-
activation with these dipeptide substrates is substantially
the same when the electrostatic interaction is negligible.
Thermolysin [EC 3.4.24.27] is a thermostable neutral
metalloproteinase isolated from Bacillus thermoproteo-
1)
lyticus. It requires essentially one zinc ion for enzyme
2
)
activity and four calcium ions for structural stability.
We have previously reported remarkable activation of
thermolysin by high concentrations (1–5 M) of neutral
salts in the hydrolysis and synthesis of ZDFM, a
3
)
precursor of a synthetic sweetener, aspartame, and in
4
,5)
the hydrolysis of FAGLA.
Activity increases appa-
rently in an exponential fashion with increases in salt
concentration, and activation is brought about solely
3
,4)
through an increase in kcat.
This suggests that
activation is attributable to stabilization of the transition
state of the enzymatic reaction. Little is known,
however, about the cause of salt-activation of thermo-
lysin at present.
In this study, we describe salt-activation of thermo-
lysin using a positively charged fluorescent substrate,
MOCAc-PLGL(Dpa)AR. The peptide bond between the
6
)
Gly and Leu residues was cleaved by thermolysin, and
the amount of the product MOCAc-PLG was estimated
by fluorescence intensity at 393 nm with excitation at
On the other hand, the v =vo value obtained with
x
MOCAc-PLGL(Dpa)AR at x > 0:5 is much higher than
those with FAGLA and ZDFM, suggesting that activa-
tion at x > 0:5 depends on the chain length or hydro-
phobicity of the substrate. The hydrolysis of MOCAc-
PLGL(Dpa)AR by MMP-7 is activated by NaCl, and
this activation is brought about solely through an
3
28 nm by comparing it with that of the authentic
MOCAc-PLG solution. The reaction was carried out
under pseudo-first-order conditions, where the substrate
concentration [S] is much lower than the Km value, in
7
)
7)
order to avoid absorptive quenching effects. Thus the
decrease in Km. The exponential behavior of salt-
y
To whom correspondence should be addressed. Tel: +81-75-753-6266; E-mail: inouye@kais.kyoto-u.ac.jp
Abbreviations: FAGLA, N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH2; MES, 2-(N-morpholino)ethanesulfonic acid; MMP-7, matrix metalloproteinase
7
; MOCAc-PLG, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly; MOCAc-PLGL(Dpa)-AR, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-
3
Leu-[N -(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2; ZDFM, N-carbozoxy-L-Asp-L-Phe-methyl ester

1812
H. ONEDA et al.
Fig. 1. Effect of NaCl on the Thermolysin-catalyzed Hydrolysis of MOCAc-PLGL(Dpa)AR.
ꢁ
The reaction was performed in 50 mM Tris–HCl buffer (pH 7.5) plus 10 mM CaCl2, 0.6% dimethyl sulfoxide at 25 C. The initial
concentrations of thermolysin and MOCAc-PLGL(Dpa)AR were 7.2 nM and 750 nM respectively. A, effect of NaCl on the reaction rate (v) and
the degree of activation; B, logarithmic relationship of the reaction rate with [NaCl].
Fig. 2. pH-Dependence of the Thermolysin-catalyzed Hydrolysis of MOCAc-PLGL(Dpa)AR.
The reaction was performed in 50 mM acetate (pH 4.0–5.8), MES (pH 5.8–7.0), Tris–HCl (pH 7.0–9.0) plus 10 mM CaCl2, 0.6% dimethyl
ꢁ
sulfoxide at 25 C. NaCl concentrations added to the buffers: 4 M,
PLGL(Dpa)AR were 7.2 nM and 750 nM respectively. A, effect of pH on the reaction rate (v); B, logarithmic relationship of the reaction rate
; 0 M,
. The initial concentrations of thermolysin and MOCAc-
with pH.
enzyme and substrate.
The pH-dependence of the thermolysin-catalyzed
hydrolysis of MOCAc-PLGL(Dpa)AR was also exam-
ined. A narrow bell-shaped pH-dependence of thermo-
lysin activity was obtained in the absence and the
Table 1. Degree of Activation of Thermolysin and MMP-7 by NaCl
Substrate
Thermolysin
MMP-7a
FAGLAb
1:9
1:2 (x < 0:5)
1
xc
n.d.d
n.d.
ZDFMe
x
x
:8 (x > 0:5)
x
presence of 4 M NaCl. The maximum activities were
ꢀ1
x
MOCAc-PLGL
-(Dpa)AR
4:7 (x < 0:5)
x
2:1 (x < 0:5)
x
0.59 and 22.7 nM s respectively (Fig. 2A). The pH-
2:3 (x > 0:5)
1:4 (x > 0:5)
dependence of v follows the ionization of free thermo-
lysin under the pseudo-first-order conditions. The pKe1
and pKe2 values were determined by the log v vs. pH
plots (Dixon plots) to be 5.56 and 7.52 in the absence of
NaCl, and 6.05 and 7.54 in the presence of 4 M NaCl
respectively (Fig. 2B). These values were separated by
less than 2 pH units, and thus the pKe values were
evaluated also according to the following equation using
a, Ref. 7; b, Ref. 4; c, Degree of activation at x M NaCl at pH 7.5, 25 C; d,
not detected; e, Ref. 3.
ꢁ
activation of MMP-7 is similar to that of thermolysin,
but the magnitude of the activation is much lower and
the activation is attributable to stabilization of the ES
complex. These observations suggest that halophilic
properties are different depending on the species of
8
)
the non-linear least-squares method:

Salt-activation of Thermolysin
bacteria (in Japanese). J. Ferment. Technol., 40, 346–353
1962).
1813
þ
þ
v ¼ vmax=½1 þ ð½H ꢃ=Ke1Þ þ ðKe2=½H ꢃÞꢃ
ð1Þ
(
2
)
Latt, S. A., Holmquist, B., and Vallee, B. L., Thermoly-
sin: A zinc metalloenzyme. Biochem. Biophys. Res.
Commun., 37, 333–339 (1969).
Inouye, K., Effects of salts on thermolysin: Activation of
hydroysis and synthesis of N-carbozoxy-L-aspartyl-L-
phenylalanine methyl ester, a unique change in the
absorption spectrum of thermolysin. J. Biochem., 112,
335–340 (1992).
where vmax is the theoretical maximal activity. vmax,
ꢀ
and 7.45 at 0 M NaCl, and 32.9 nM s 1, 6.12, and 7.40 at
pKe1, and pKe2 were determined to be 0.80 nM s 1, 5.60,
ꢀ
3
)
4
M NaCl, respectively. There is no significant difference
in pKe values between the two methods, and it is noted
that pKe1 shifts from 5.6 to 6.1 with an increase in
[
NaCl] from 0 to 4 M, while pKe2 is unaltered. This shift
can be attributed to a microenvironmental change
around the pKe1 group, suggesting that high concen-
trations of NaCl may induce a conformational change in
thermolysin. These observations might provide a clue to
understand salt-activation of thermolysin, MMP-7, and
other halophilic enzymes.
4) Inouye, K., Lee, S.-B., and Tonomura, B., Effect of amino
acid residues at the cleavable site of substrates on the
remarkable activation of thermolysin by salts. Biochem.
J., 315, 133–138 (1996).
5
)
Inouye, K., Lee, S.-B., Nambu, K., and Tonomura, B.,
Effects of pH, temperature, and alcohols on the remark-
able activation of thermolysin by salts. J. Biochem., 122,
3
58–364 (1997).
Acknowledgments
6
7
)
)
Muta, Y., and Inouye, K., Inhibitory effects of alcohols on
thermolysin activity as examined using a fluorescent
substrate. J. Biochem., 132, 945–951 (2002).
Oneda, H., and Inouye, K., Effects of dimethyl sulfoxide,
temperature, and sodium chloride on the activity of
human matrix metalloproteinase 7 (matrilysin). J. Bio-
chem., 128, 785–791 (2000).
This study was supported in part by grants (nos. 0150
and 0345, to K.I.) from the Salt Science Foundation of
Tokyo.
References
8
)
Segel, I. H., ‘‘Enzyme Kinetics’’, John Wiley and Sons,
New York, pp. 914–917 (1975).
1)
Endo, S., Studies on protease produced by thermophilic