Temperature and salts effects on the peptidase activities
of the recombinant metallooligopeptidases neurolysin
and thimet oligopeptidase
Vitor Oliveira
1
, Reynaldo Gatti
2
, Vanessa Rioli
3
, Emer S. Ferro
3
, Alberto Spisni
2,4
, Antonio C. M. Camargo
5
,
Maria A. Juliano
1
and Luiz Juliano
1
1
Department of Biophysics, Escola Paulista de Medicina, Sa
˜
o Paulo, Brazil;
2
Centro de Biologia Molecular Estrutural, National
Laboratory of Synchrotron Light (CBME-LNLS), Campinas, Brazil;
3
Department of Histology, Institute of Biomedical Sciences,
Universidade de Sa
˜

Na
2
SO
4
. Concentration higher than 0.2 N NH
4
Cl and NaI
reduced TOP activity, while 0.5 N or higher concentration
of NaCl, KCl and Na
2
SO
4
increased TOP activity. Neu-
rolysin was strongly activated by NaCl, KCl and Na
2
SO
4
,
while NH
4
Cl and NaI have very modest effect. High
positive values of enthalpy (DH*) and entropy (DS*) of
activation were found together with an unusual tempera-
ture dependence upon the hydrolysis of the substrates.
The effects of low temperature and high NaCl concen-
tration on the hydrolytic activities of neurolysin and TOP
do not seem to be a consequence of large secondary
structure variation of the proteins, as indicated by the far-
UV CD spectra. However, the modulation of the activities
of the two oligopeptidases could be related to variations

Efficient oligopeptidases are required to metabolize
biologically active peptides before and after their interac-
tion with cell receptors, this is particularly relevant with
neuropeptides that lack classical reuptake mechanisms for
recycling components into the cell. TOP exhibits charac-
teristics of both metabolizing and processing enzymes,
and has multiple peptide substrates as GnRH [17],
neurotensin [18], bradykinin [19], somatostatin 1–14 [20],
and nociceptin [21]. TOP also processes Met- and
Correspondence to L. Juliano, Departamento de Biofisica, Escola
Paulista de Medicina, Rua Treˆ s de Maio, 100, 04044-020 Sa
˜
oPaulo,
SP, Brazil. Fax: + 55 11 5575 9040, Tel.: + 55 11 5575 9617,
E-mail:
Abbreviations:Abz,ortho-aminobenzoic acid; EDDnp,
N-(2,4-dinitrophenyl) ethylenediamine; IQF peptide, internally
quenched fluorescent peptide, TOP, thimet oligopeptidase.
Enzymes: thimet oligopeptidase (TOP, EC 3.4.24.15); neurolysin
(EC 3.4.24.16).
*Present address: Department of Experimental Medicine, University
of Parma, Parma, 43100, Italy.
(Received 10 April 2002, revised 3 July 2002, accepted 19 July 2002)
Eur. J. Biochem. 269, 4326–4334 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03129.x
Leu-enkephalin from the enkephalin-containing peptides
[22], and the specific TOP inhibition increased Met-enke-
phalin antinociception in rodents [23]. TOP and neurolysin
are able to hydrolyze the biologically active peptide
neurotensin (NT) in vitro and they could be participating
in the catabolism of this biologically active peptide. In vivo

In the present work we report the neurolysin and TOP
hydrolytic activities towards IQF peptides derived from
Abz-GGFLRRVQ-EDDnp [Abz, ortho-aminobenzoic
acid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine], in
which Val was substituted by 11 different natural amino
acids. This sequence was chosen because we have previously
observed efficient hydrolysis by TOP of the peptide Abz-
GGFLRRV-EDDnp at L-R bond and the addition of Gln
at C-terminal site (Abz-GGFLRRVQ-EDDnp) resulted in
two cleavages, at L–R and or at R–R bond. We made
modifications at Val position in order to verify the influence
of the nature of the amino acid at this position on
determination of the cleavage sites and on the amount of
their hydrolysis. This series of peptides was chosen because
the P
2
¢ and P
3
¢ positions were demonstrated to be very
determinant on the specificity of neurolysin and TOP [5].
The kinetic parameters of hydrolysis and the variations of
the cleavage sites of this series of peptides by these two
oligopeptidases were evaluated in different conditions of
temperature and salts.
MATERIALS AND METHODS
Thimet oligopeptidase (TOP)
The purified recombinant rat testes TOP (rTOP) was
obtained as previously described [33]. Details about the
procedures applied for enzyme characterization and active
site titration were described elsewhere [5].

with a 10 (or 30))50 (or 60)%
gradient of solvent B over 30 or 45 min. Analytical HPLC
was performed using a binary HPLC system from Shima-
dzu with a SPD-10AV Shimadzu uv-vis detector and a
Shimadzu RF-535 fluorescence detector, coupled to an
Ultrasphere C-18 column (5l,4.6· 150 mm) which was
eluted with solvent systems A and B at a flow rate of
1mLÆmin
)1
and a 10–80% gradient of B over 20 min. The
HPLC column eluates were monitored by their absorbance
at 220 nm and by fluorescence emission at 420 nm follow-
ing excitation at 320 nm. The molecular mass and purity of
synthesized peptides were checked by MALDI-TOF mass
spectrometry (TofSpec-E, Micromass) and/or peptide
sequencing using a protein sequencer PPSQ-23 (Shimadzu
Tokyo, Japan).
Kinetic assays
The Michaelis parameters were determined by initial rate
measurements. The hydrolysis of the fluorogenic peptidyl
substrates at 37 °Cin50m
M
Tris/HCl buffer pH 7.4
containing 100 m
M
NaCl was followed by measuring the
fluorescence at k
em
¼ 420 nm and k
ex

as by using Eadie–Hofstee plots. All the obtained data were
fitted to nonlinear least square equations, using
GRAFIT
v3.0
from Erithacus Software [38].
The hydrolysis of the substrates cleaved at two peptide
bonds by TOP and neurolysin can be represented as shown
in Scheme 1, whose equation for velocity is Eqn (1). V
t
is
the sum of the velocities of formation of the products (P
a
and P
b
). V
a
max
is kp
a
· [E] and V
b
max
is kp
b
· [E], and [E] is
the total enzyme concentration in the assay. All the obtained
data with the peptides cleaved at two bonds fitted to
nonlinear least square plot of Eqn (1). The overall V
max
was

determined under first-order conditions, we used substrates
concentrations 10-fold less than K
m
. The obtained first-
order rate constants were divided by the total enzyme
concentration to provide k
cat
/K
m
. As the products Abz-
GGFL, Abz-GGFLR and their respective C-terminal
fragments were resistant to hydrolysis by TOP, and the
products Abz-GGFLR, Abz-GGFLRR and their respect-
ive C-terminal fragments were also resistant to hydrolysis by
neurolysin, we could determine the specificity rate constants
(k
cat
/K
m
) under first-order conditions, even for the peptides
hydrolyzed at two peptide bonds. This procedure was used
in the assays conduced at different temperatures and at
different salt concentrations [5].
Temperature dependence of the hydrolysis reaction
rates of the substrates by neurolysisn and TOP
The temperature dependence of the rate constants was
determined in thermostated cell holders. The reactions
were started after the thermal equilibrium had been
reached in the cell. Typically the reactions were carried
out in 1 mL of 50 m

R
N
A
h

þ
DS
Ã
R
À
DH
Ã
RT
ð2Þ
DG
Ã
¼ DH
Ã
À TDS
Ã
ð3Þ
Dependence of the hydrolysis reaction rates
by neurolysisn and TOP on concentration and chemical
nature of salts
The dependence of the rate constants according to the
concentration and the chemical nature of salts were
determined in 1 mL of 50 m
M
Tris/HCl buffer pH 7.4
containing different concentrations of NaCl, KCl, NH

solution of recrystalized d-10 camphorsulphonic acid.
Ellipticity is reported as mean residue molar ellipticity, [h]
(degÆcm
2
Ædmol
)1
). The spectrometer conditions were typi-
cally: spectral range 195–260 nm, 100 mdeg sensibility;
0.2 nm resolution; 4 s response time; 20 nmÆmin
)1
scan rate,
7 accumulations at the appropriate temperature (10, 25 or
37 °C). The 100 mdeg sensibility is used in our routine that
leads to the lower noise-signal relationship. The control
baseline was obtained with solvent and all the components
without the proteins. All the data were obtained with three
Scheme 1.
4328 V. Oliveira et al. (Eur. J. Biochem. 269) Ó FEBS 2002
different solutions of the proteins. The quality of data was
certified by the correspondence of the amount of secondary
structures obtained by CD data deconvolution with those
from the 3D structure of neurolysin. The errors of
prediction on the range 195–260 nm and 200–260 nm were
5% using the
CDNN
program [40].
RESULTS
Kinetic parameters for the hydrolysis of IQF peptide
series Abz-GGFLRRXQ-EDDnp by TOP and neurolysin
Table 1 shows the kinetic parameters of the hydrolysis by

m
values obtained with
TOP were for substrates I and II containing Arg and His at
the X position, respectively.
Neurolysin, like TOP, hydrolyzed all the substrates at
R–R bond, but the alternative cleavage site was at R–X
bond in peptides II to IV, VII, IX and X, which contain at
the X position of the series Abz-GGFLRRXQ-EDDnp
essentially hydrophobic amino acids. The peptides hydro-
lyzed exclusively at R–R bonds contain at the X position
charged amino acids (Arg, Asp and Glu) or amino acids
with small hydrophobic side chain (Ala, Pro and Val). The
highest k
cat
/K
m
value for neurolysin was observed with
the hydrolysis of the substrate with Ala (peptide VI), and the
lowest k
cat
/K
m
values was the peptide with Asp (peptide
XII) at the X position.
Temperature dependence of the substrate hydrolysis
by TOP and neurolysin
The preference of cleavage at the L–R or R–R bond for
TOP and at the R–R or R–X bond for neurolysin in the
case of the substrates containing Val, Asp and Ile at the
X position in the studied series were determined at

m
¼ (k
a
cat
+ k
b
cat
)/K
m
. L–R, R–R and R–X indicate the cleavage sites. Qf 7 is the abbreviation used for the
peptide Abz-GGFLRRV-EDDnp. The kinetic experiments were conduced at 37 °Cin50m
M
Tris/HCl buffer containing 0.1
M
NaCl. For XIII,
cleavage site is L–R.
Number X
TOP (24.15) Neurolysin (24.16)
k
cat
(s
)1
)
K
m
(l
M
)
k
cat

II H – 10 1.4 7.1 1.6 0.6 1.7 1.3
III Y – 19 4.3 4.4 0.3 0.2 0.6 0.8
IV F – 15 3.6 4.2 0.2 0.3 0.7 0.7
V P – 7.4 2.2 3.4 1.8 – 1.2 1.5
VI A – 15 4.8 3.1 3.2 – 0.5 6.4
VII N 1.6 5.5 1.8 3.9 0.6 0.3 1.5 0.6
VIII V 1.7 2.8 1.7 2.6 1.4 – 2.1 0.7
IX I 0.8 4.3 2.0 2.5 0.7 0.1 1.8 0.4
X L 1.8 3.5 2.5 2.1 0.2 0.5 0.7 1.0
XI E 0.7 2.2 1.5 1.9 1.1 – 1.9 0.6
XII D 3.4 1.6 3.2 1.6 1.0 – 3.3 0.3
XIII Qf 7 0.7 – 1.7 0.4 2.0 – 2.2 0.9
Ó FEBS 2002 Modulation of thimet oligopeptidase and neurolysin activities (Eur. J. Biochem. 269) 4329
Abz-GGFLRRXQ-EDDnp containing Val, Ile and Ala at
the X position. The peptide containing Asp were only
assayed with TOP. Linear Eyring plots (ln[k/T ]vs.1/T )
were obtained for the hydrolysis of Qf 7 by both enzymes
and for the hydrolysis of the substrate containing Val by
neurolysin. The Eyring plots for the hydrolysis by TOP of
the peptides containing Ile, Val and Asp deviated from the
linearity above 25 °C. The plot for the reaction of the
peptide containing Ala with TOP was not linear in all
studied range of temperature (Fig. 1A). The Eyring plots
obtained for neurolysin reactions with the peptides
containing Ala and Ile at the X position gave two linear
fittings, above and below % 22 °C (Fig. 1B), indicating
different rate-limiting steps at each temperature range
(above and below 22 °C).
The DG* DH*andDS* values were taken from Eyring
plots and are shown in Table 3. In addition to the

activities on Qf7 were also determined in the presence of
2
M
NaCl. In this condition, the Eyring plots obtained for
TOP and neurolysin gave two linear fittings, above and
below % 22 °C, which contrast with linear plots obtained in
the absence of salt. Similar to all others, substrates with
similar temperature behavior resulted in DH*andDS*
values at temperature range 25–37 °C significantly lower
than those at 10–20 °C(Table3).
Influence of the chemical nature of salts on the TOP
and neurolysin activities
Using Qf7 as a reference substrate, which was hydrolyzed
only at L–R bond, we studied the effects of different salts on
TOP and neurolysin activities. The results are shown in
Fig. 2A,B, respectively. TOP was activated by all the
assayed salts (NaCl, KCl, Na
2
SO
4
,NH
4
Cl and NaI) at
low concentrations. However, the increase of NH
4
Cl or NaI
concentrations reduced TOP activity, in contrast to NaCl,
KCl and Na
2
SO

Tris/HCl buffer con-
taining 0.1
M
NaCl.
T °C
Cleaved bond %, TOP
X ¼ V X ¼ D X ¼ I
L–R R–R L–R R–R L–R R–R
10 18 82 39 61 11 89
20 26 74 50 50 9 91
30 27 73 59 41 11 89
37 38 62 64 36 17 83
4330 V. Oliveira et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Circular dichroism spectra of TOP and neurolysin
The CD spectra of TOP and neurolysin show a predomi-
nance of a helical structures as shown in Fig. 3 (without
smoothing and curve fitting). For neurolysin spectra, the
results obtained in the deconvolution of the CD data, using
the
CDNN
program [40], are consistent with the helix content
found in the neurolysin crystal structure, as the helix content
from the crystal structure was 53% [3]; and from the
deconvolution of CD at 37 °C in the absence of NaCl (195–
260 nm) was 51%. For the spectrum of TOP in the same
conditions the deconvolution indicated 45% of a helix
content. This result is close to that of neurolysin and
consistent with consensus secondary structure prediction
obtained from different algorithms (
DPM

repetitive sequences of tri-peptides [42] by TOP three or four
amino-acid residues from the C-terminal end of the
substrates. TOP and neurolysin also hydrolyze IQF peptides
derived from bradykinin by a similar way. In this case,
depending on the sequence and size of the substrates, the
hydrolysis were observed 6–10 amino acids from the
C-terminal end of the peptides but with very low efficien-
cies [5]. Comparatively, neurolysin hydrolyzed closer to
C-terminal end than TOP the series Abz-GGFLRRXQ-
EDDnp, as also observed for hydrolysis of neurotensin
derivatives [6]. In fact, despite the R–R bond being the
Table 3. Activation parameters for TOP and neurolysin reactions with the substrates of the series Abz-GGFLRRXQ-EDDnp and Abz-GGFLRRV-
EDDnp (Qf 7). The kinetic experiments were conduced in 50 m
M
Tris/HCl buffer containing 0.1
M
NaCl. The parameters were calculated as mean
value ± S.D.
Enzyme Substrate
Temperature
Range
a
°C
DG*
kJÆmol
)1b
DH*
kJÆmol
)1
DS*

Table 4. Influence of NaCl concentration on the specificity constant (k
cat
/K
m
) for hydrolysis of peptides derived from Abz-GGFLRRXQ-EDDnp by
TOP and neurolysin. The unit of k
cat
/K
m
is l
M
)1
Æs
)1
. The effect of NaCl on the variation of cleavage sites for neurolysin was perfomed only for the
substrate containing Ile. The kinetic experiments were conduced at 37 °Cin50m
M
Tris/HCl pH 7.4.
NaCl (
M
)
X=A
k
cat
/K
m
X=V
a
k
cat

GGFLRRXQ-EDDnp, TOP also hydrolyzed the L–R
bond while neurolysin hydrolyzed the R–X bond. These
observations are in accordance with the hydrolysis by
recombinant neurolysin and TOP of natural substrates,
such as bradykinin, neurotensin, metorphinamide, dynor-
phin A 1–8 and angiotensin I [34].
The 3D structure of rat neurolysin [3] demonstrated that
the substrate binding site is a channel, which amino-acid
chains of its wall are connected by flexible loops and open
coil regions. As a consequence, it is tempting to speculate
that these flexible structures in neurolysin and TOP can
accommodate peptides inside their substrate binding chan-
nels with different degree of restrictions, which could be
responsible for the absence of specificity, particularly for S
1
subsite. The displacement of the cleavage site to R–R bond
on the hydrolysis of the substrates Abz-GGFLRRXQ-
EDDnp at low temperatures and at high NaCl concentra-
tions (Tables 2 and 4) suggested modifications on the
channel binding site of neurolysin and TOP, better accom-
modating the R–R residues for hydrolysis. In addition, the
kinetic parameters k
cat
/K
m
varied with NaCl concentration,
and the extent of it was dependent on the substrate
(Table 4) and on the nature of the salts (Fig. 2). Therefore,
the structures of TOP and neurolysin could be changed by
salts, as a similar manner as the recently described activation

cat
/K
m
value obtained in a determined salt concentration.
The concentration is presented in normality (N).
Fig. 3. CD spectra of TOP and neurolysin collected at 37 °CinTris
50 m
M
pH 7.4 in the absence (full circles) or in the presence of 1
M
NaCl
(open triangles). These data are without curve fitting or smoothing.
4332 V. Oliveira et al. (Eur. J. Biochem. 269) Ó FEBS 2002
detectable changes in their secondary structures. The
intrinsic mechanism of activation of TOP and neurolysin
by salts was not determined, however, change of rate-
limiting step or the speed up of isomerization of the enzyme-
substrate complex were described for other peptidases
[44,45]. At low salt concentrations the predominant effects
seems to be due to the shielding of charges present in the
enzymes and in the substrates, as suggested the results
obtained with the peptide containing X ¼ Asp (Table 4).
Finally the activation of TOP and neurolysin by increasing
concentration of NaCl was also verified with the substrate
Abz-GFSPFIQ-EDDnp, which does not have charged side
chains (results not shown) indicating that NaCl affects TOP
and neurolysin structures.
It is noteworthy the unusual temperature dependence of
the k
cat

alterations in the enzyme structure with the temperature. In
the temperature experiments with neurolysin and TOP,
where a break in the Eyring plots occurs showing two linear
sections can be interpreted as two different rate limiting
steps at each temperature range [46]. Similar unusual
temperature dependence of the k
cat
/K
m
ratio according with
the substrate was observed for the oligopeptidase B [47].
The activation parameters were estimated for the tem-
perature ranges in which linear Eyring plots were obtained
(Table 3). In all reactions for both TOP and neurolysin
markedly positive enthalpies and entropies of activation
were verified. Positive entropy of activation can be associ-
ated with reorganization of the protein structure, which may
involve an unfolding process or changes in the water layer
around the reactants [46]. On the other hand, as the
substrates must go inside a channel to find the catalytic
machinery, a considerable amount of water should be lost
from the substrates, and in this case the entropy contribu-
tion will be positive. This interpretation could be relevant
considering the activities of neurolysin and TOP on
neuropeptides containing free or amidated C-terminal
carboxyl group, as more water should be organized around
to the free C-terminal carboxyl group and the hydrolysis of
these substrates should be more sensitive to changes of
temperature and ionic strength. Finally, differences in the
specificities and effects of salts and temperature between the

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