Structural and Functional Peculiarities of Homologous Domains of Angiotensin-Converting Enzyme

Article abstract of DOI:10.1023/A:1026045324442

Somatic angiotensin-converting enzyme (ACE) consists of two homologous domains, each of them containing an active site. Differences in substrate specificities and affinity to inhibitors of the active sites of the two domains of bovine ACE are described. The ACE domains demonstrate different thermostability, and the reasons for this difference are analyzed. A structural model of the ACE domains is suggested, which allows us to reveal the structural subdomain important for the protein stability and localize the hydrophobic and carbohydrate-binding sites.

Full text of DOI:10.1023/A:1026045324442

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 5, 2003, pp. 426–433. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 5, 2003, pp. 470–478.
Original Russian Text Copyright © 2003 by Voronov, Binevski, Zueva, Palyulin, Baskin, Orlova, Kost.
Structural and Functional Peculiarities of Homologous Domains
of Angiotensin-Converting Enzyme
S. V. Voronov, P. V. Binevski, N. A. Zueva, V. A. Palyulin, I. I. Baskin,
M. A. Orlova, and O. A. Kost1
Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia
Received December 20, 2002; in final form, January 22, 2003
Abstract—Somatic angiotensin-converting enzyme (ACE) consists of two homologous domains, each of them
containing an active site. Differences in substrate specificities and affinity to inhibitors of the active sites of the
two domains of bovine ACE are described. The ACE domains demonstrate different thermostability, and the
reasons for this difference are analyzed. A structural model of the ACE domains is suggested, which allows us
to reveal the structural subdomain important for the protein stability and localize the hydrophobic and carbo-
hydrate-binding sites.
Key words: angiotensin-converting enzyme, secondary structure, three-dimensional structure; 8-anilino-1-
naphthalenesulfonic acid; thermal inactivation; γ-inactivation
1
INTRODUCTION
The three-dimensional structure of the full-size
ACE and its domains is still not determined, and the
Angiotensin-converting enzyme (peptidyldipepti- structural backgrounds of the functional peculiarities of
dase A, EC 3.4.15.1) is a zinc-containing metallopro- ACE domains are unknown.
tease, which consists of 1277 amino acid residues and
2
The ACE catalytic sites hydrolyze the same peptide
substrates and bind the same inhibitors. However, these
sites are distinguished in the rate of the peptide hydrol-
ysis, efficiency of the inhibitor binding, and the profile
of activation by chloride anions [2]. It has previously
been assumed that catalytic hydrolysis proceeded inde-
pendently on the two ACE active sites [7]. However,
some information now appeared that the active sites of
the enzyme interact with each other [6, 8, 9], which
implies a spatial proximity and contacts between the
domains.
belongs to the zincin class. The unique feature of the
enzyme molecule synthesized in somatic cells is the
presence of two highly homologous domains within a
single polypeptide chain, each domain containing its
own active site [1]. These domains are called as N-
domain and C-domain according to their location in the
polypeptide chain. Their homology is as high as 67–
7
3% (depending on an organism), and the identity of
amino acid residues in their central parts containing the
zinc-binding motifs reaches 89% [2]. At the same time,
both domains themselves and their active sites are not
identical. For example, the N-terminal domain of
In this study, we present for the first time the infor-
human ACE was shown to be much more glycosylated mation on the substrate specificity of bovine ACE and
than its C-domain [3]. In the experiments with mutant the affinity of its active sites to the inhibitors, on the dif-
ferent stability of its two enzymic domains and its pos-
sible reasons, and suggest a structural model of the
ACE domains.
forms of human ACE, the recombinant N-domain was
found to be more stable at high temperatures than the
C-domain or the two-domain form of ACE [4]. Similar
results were also obtained for the bovine ACE by the
method of differential scanning calorimetry. The
somatic enzyme had two peaks of thermal absorption at
RESULTS AND DISCUSSION
5
5 and 70°ë, whereas the individual N-domain of ACE
It is known that a partial denaturation of the somatic
enzyme results in the denaturation of its C-domain,
whereas the N-domain preserves its catalytic activity
and can be isolated in the individual form after the lim-
ited proteolysis of the denatured protein [10]. The
bovine testicular enzyme was used as an individual
C-domain. This enzyme is identical to the C-domain
of the somatic ACE except for short N-terminal
sequence that does not influence the enzyme catalytic
prepared by a limited proteolysis of the parent somatic
form exhibited only one high-temperature peak [5].
The N-domain is also more stable toward the action of
urea and the extreme pH values [6].
1
Corresponding author; fax: +7 (095) 939-5417; e-mail:
kost@enzyme.chem.msu.ru
Abbreviations: ACE, angiotensin-converting enzyme; ANS, 8-
anilino-1-naphthalenesulfonic acid; and FA, 2-furylacryloyl.
2
1
068-1620/03/2905-0426$25.00 © 2003 MAIK “Nauka /Interperiodica”

STRUCTURAL AND FUNCTIONAL PECULIARITIES
427
Table 1. Kinetic parameters of the hydrolysis of the synthetic tripeptide substrates by the single-domain forms of the bovine
ACE*
N-domain of ACE
Testicular ACE
Substrate
–
1
–1
k , s
K , mM
k , s
K , mM
m
cat
m
cat
FA-Phe-Gly-Gly
Hip-His-Leu
279 ± 10
12 ± 1
1.40 ± 0.02
0.50 ± 0.05
0.15 ± 0.02
0.05 ± 0.02
0.14 ± 0.01
0.05 ± 0.01
260 ± 14
12 ± 1
0.50 ± 0.02
0.60 ± 0.03
0.13 ± 0.02
0.05 ± 0.01
0.17 ± 0.02
0.12 ± 0.02
Cbz-Phe-His-Leu
FA-Phe-Ala-Ala
FA-Phe-Ala-Lys
FA-Phe-Phe-Arg
122 ± 10
35 ± 3
19 ± 2
178 ± 15
85 ± 7
35 ± 3
45 ± 4
76 ± 8
*
The substrates were hydrolyzed in 50 mM Hepes buffer (pH 7.5) containing 150 mM NaCl and 1 µM ZnCl at 25°C.
2
functions [11]. Thus, we could compare the catalytic mal denaturation of the somatic ACE in a wide range of
functions of two individual domains of bovine ACE.
temperatures and the determination of the values of
activation energy for the denaturation of each domain
in the enzyme. The data are plotted in the Arrhenius
coordinates in Fig. 1b. A more than double difference in
Kinetic parameters of the hydrolysis of synthetic
tripeptide substrates by the individual active sites of
bovine ACE are given in Table 1. It was found that a
number of substrates were equally hydrolyzed on both
active sites of bovine enzyme (for comparison note that
the C-domain of human ACE hydrolyzes Hip-His-Leu
substrate nine times faster than the N-domain [7]). The
substrates FA-Phe-Ala-Ala, FA-Phe-Ala-Lys and FA-
Phe-Ala-Arg were faster hydrolyzed by the C-domain
the E values illustrates the difference in thermostabil-
‡
ity of the homologous domains of ACE.
It is important that the different thermostability of
the domains opens a possibility for the selective dena-
turation of one of the domains (C-domain) with the for-
mation of the catalytically active individual N-domain
(differences in the values of the catalytic constants
(after proteolysis) or the N-domain within the full-size
along with the similar values of the Michaelis con-
stants), but Cbz-Phe-His-Leu was faster hydrolyzed on
the N-domain of ACE (whereas the human ACE hydro-
lyzed this substrate with the practically equal rate on
both sites [12]). As a whole, these data agree with the
earlier proposal [7, 13] about lesser demands of the C-
domain active site to the structure of substrate.
enzyme with the denatured C-domain; in this case, the
presence of denatured C-domain had no effect either on
the stability or on the catalytic functions of the N-
domain [5]. Thus, we can conclude that the domains in
the molecule of somatic ACE are denatured indepen-
dently of each other, which suggests that the spatial
structures of these domains are also independent.
The efficiencies of inhibition of both active sites of
bovine ACE by the reversible competitive inhibitors are
A higher stability of the ACE N-domain was also
given in Table 2. The only significant difference in the demonstrated in the experiments of the enzyme dena-
values of inhibition constants was observed for capto- turation by action of the products of water radiolysis
pril that more effectively inhibited the N-domain of the with γ-irradiation. In contrast to the heat treatment that
enzyme as in the case of human ACE [14].
involves the whole volume of the molecule, the prod-
ucts of water radiolysis mainly react with the protein
surface [15]. It was found that the individual N-domain
Thus, the bovine ACE active sites somewhat differ
in their substrate specificity and in their affinity to
inhibitors, and this differences does not coincide with
those previously revealed for the active sites of the
human enzyme.
Table 2. Inhibition constants of hydrolysis of Cbz-Phe-His-
Leu by single-domain ACE forms by competitive inhibitors
Functional difference between the enzyme domains
is also revealed when comparing their stability. The
kinetics of the irreversible thermal denaturation of the
two-domain somatic ACE has a pronounced two-phase
character (Fig. 1a), which does not depend on the pro- Lisinopril
K , M
i
Inhibitor
N-domain
testicular ACE
(2.0 ± 0.1) × 10–10
(5.0 ± 0.2) × 10–10
(1.2 ± 0.2) × 10–10
(9.1 ± 0.9) × 10–9
tein concentration and suggests the presence in the sys-
tem of at least two fractions with different stability. The
rate constant of the thermal denaturation of the testicu-
lar ACE coincided with that of the labile fraction,
whereas the stable fraction was denatured with the
same rate constant as the isolated individual N-domain.
The results allowed analysis of the kinetics of the ther-
Captopril
PP-09
(1.5 ± 0.2) × 10–8
(8.6 ± 0.4) × 10–8
(4.7 ± 0.4) × 10–8
(2.6 ± 0.1) × 10–7
Enalapril
maleate
Note: The hydrolysis was performed in 50 mM Hepes buffer (pH
7.5) containing 150 mM NaCl and 1 µM ZnCl2 at 37°C.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

4
28
VORONOV et al.
Residual activity, %
00
Residual activity, %
100
1
(
‡)
–
–
–
0.5
1.0
1.5
2
8
6
4
2
0
0
0
0
8
0
60
4
3
0
20
40
60
Time, min
4
2
0
0
1
0
10
20
30
40
50
60
0
50
100
150 D, Gy
Time, min
ln(kin)
Fig. 2. The dependence of the residual activity of (1, 2)
somaticACE and (3, 4)ACE N-terminal domain on the dose
of γ-irradiation (1, 3) in the absence and (2, 4) in the pres-
ence of ANS at the concentration of 50 mM in the mixture
of 0.025 M phosphate buffer–0.025 M borate buffer
(
b)
–
–
–
–
–
5
6
7
8
9
+
2+
E = 120 – 10 kJ/mol
a
(pH 6.5) containing 0.15 M NaCl and 1 µM Zn . ACE con-
centration 10 nM.
2
The individual N-domain turned out to be insignifi-
cantly stabilized in the presence of ANS (Fig. 2), which
suggests that the single hydrophobic protector-binding
site is in the C-domain of the enzyme. We should stress
that the molecule of somaticACE sensitive to radiolysis
became much more stable after the formation of ANS
complex than its initially more stable N-domain (see
curves 2 and 3 in Fig. 2). These results point out to the
existence of contacts between the enzyme domains,
which lead to the mutual influence of the domains,
including the stability toward radiolysis.
We began the analysis of possible structural differ-
ences between the domains responsible for the differ-
ences in their stability with the prediction of secondary
structure of the domains of bovine ACE on the basis of
the known amino acid sequence of the enzyme [18]. We
predicted 45% content of the α-helical and ~50% con-
tent of unordered structure; the contribution of β-struc-
tures in the form of β-turns was insignificant (no higher
than 5%). According to CD spectroscopy, 37% α-helix
content is in the bovine ACE, while β-structure content
is insignificant [5].
+
1
E = 258 – 30 kJ/mol
a
2
.95
3.00
3.05
1
3.10
3
–1
/T × 10 , K
Fig. 1. The thermal inactivation of somatic ACE: (a) kinet-
ics of the thermal inactivation of ACE at 55°C (in the insert,
the same data plotted in the semi-logarithmic coordinates);
(
b) temperature dependence of the denaturation constants of
(
1) N-domain and (2) C-domain of the somatic ACE in the
mixture of 0.025 M phosphate–0.025 M borate buffer (pH
2
+
6
.5) containing 0.15 M NaCl and 1 µM Zn , expressed in
the Arrhenius coordinates.
was inactivated by the γ-irradiation more slowly than
the somatic enzyme (Fig. 2), although its specific sur-
face area is larger.
According to our model, both ACE domains have
similar secondary structures in the area of their active
sites. The most significant differences between the
domains are observed in the relatively short regions of
their N-terminal parts: they involve 1–105 aa in the N-
domain and 604–705 aa in the C-domain (Fig. 3). The
degree of homology between the N-terminal parts of N-
Radicals formed in the course of water radiolysis are
known to damage first the residues of aromatic amino
acids of a protein exposed to solvent [16]. On the other
hand, hydrophobic regions on the protein surface can
be shielded by the sorption of low-molecular com-
pounds. Previously, we have demonstrated that one
hydrophobic region capable of formation of a complex and C-domains was the lowest (49%). Two amino acid
with the ANS (a hydrophobic dye) is situated on the insertions and four sites of potential glycosylation are
ACE globule [17]. The ANS binding significantly pro- present within this area of the N-domain, whereas the
tected the somatic ACE to the action of γ-radiation, and corresponding area of the C-domain contains three
the maximum effect was achieved after the equilibrium potential glycosylation sites. Moreover, it was reported
of the complex formation was attained.
in [19] that two of these four sites in the N-domain of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

STRUCTURAL AND FUNCTIONAL PECULIARITIES
429
N–ACE
1
ELDPALQPGNFPADEAGAQIFAASFNSSAEQVLFQSTAASWAHDTNITEENARLQEE 57
C–ACE 604 MPDNYPEGIDLVSDEDEARKFVEEYDRRSQVVWNEYAEANWNYSTDISTDNSKLLME 661
N–ACE AALLSQEFSEAWGQKAKDLFDPVWQNFTDPTLLRIIGAVRTLGPANLD 105
C–ACE KNLQMANHTVKYGTWAS-KFD--VTNFQNATMKRMIKKIQDLERAALP 705
Fig. 3. The predicted secondary structures of the domains of bovine ACE in the regions of the largest structural differences between
the N-domain (1–105 aa) and the C-domain (604–705 aa). The locations of the α-helices are underlined. The potential glycosylation
sites are marked with grey boxes.
human ACE bear carbohydrate chains, whereas the cor-
Three-dimensional structures of the ACE domains
responding region of the C-domain does not contain were modeled on the basis of their primary structures,
carbohydrates. The content of α-helices in the dis- the models of their secondary structures, and the motif
cussed region of N-domain is significantly higher than of spatial folding of the neurolysin polypeptide chain as
that in the C-domain (63 and 48%, respectively). This a template (Figs. 4, 5). The model of theACE C-domain
suggests that this region of the N-domain is more con- was more complete, while that of its N-domain was
limited only to the 1–477 region. This limitation was
associated with the VVRNETH sequence (477–483 aa)
of the N-domain that is crucially distinct from the LAR-
SQDD sequence situated in the corresponding region of
formationally rigid. Moreover, the content of Pro resi-
dues, which should additionally increase the stability of
the protein, is two times higher in the N-domain region.
We can presume that a higher rigidity and a higher the C-domain (1075–1081 aa). As a result, the used
glycosylation degree of the 1–105 region of N-domain algorithm of the model design required impermissible
and a higher flexibility of the corresponding region of conformations for the side radicals of a number of
the C-domain could probably be explained by the fact amino acid residues.
that the C-domain folding proceeds under the condi-
Let’s discuss the more complete model of the C-
tions when the N-domain has already been folded at
domain in detail. Practically all of the α-helices within
least partially. An enhanced conformational flexibility
one of the ACE domains are folded in the direction
of the initial region of the N-domain is necessary for the
close to the location of the cleft containing the enzyme
compensation of the sterical limitations associated with
active site. The zinc-coordinating His959, His963, and
the presence nearby of already folded N-domain. This
Glu987 residues as well as Glu960 that participates in
flexibility possibly results in a reduced stability of the
C-domain we experimentally observed.
the polarization of the hydrolyzed peptide bond occupy
positions, which make possible the catalytic reaction.
The His1089 residue participating in stabilization of
the catalytic transition state [22] is more than 100
amino acid residues distant from these residues, which
distinguishes ACE from the other members of the zin-
cin family. In the proposed structure, this His residue
turned out to be in the common cleft with other amino
acid residues of the active site. This fact confirms the
correctness of our model. However, His1089 residue
should be significantly closer to other residues in the
real structure of the active site. Also note that His1089
is included into the long unordered loop (Fig. 4),
whereas the used algorithm for the modeling of three-
dimensional structure (like all other algorithms pres-
ently used) does not allow the determination of the con-
formation and the position of such unordered loops
with sufficient accuracy.
We made an attempt to model the three-dimensional
structure of the two ACE domains for a more detailed
description of structural differences between the
domains. At the first stage, we determined the structural
type of the protein folding allowed for each of the
domains in accordance with their primary structures
and the predicted secondary structures. It turned out
that only the structure of neurolysin (35% homology
with ACE) appeared to be close to both the N- and
C-domains of ACE among all the protein structures
known at present. Neurolysin (EC 3.4.24.16) is a zinc-
dependent metalloprotease hydrolyzing short (up to
1
7 aa) peptides, including the well-known ACE sub-
strates angiotensin and bradykinin, but their cleavage
sites differ from the sites hydrolyzed by ACE [20]. The
neurolysin polypeptide chain consists of 704 aa, which
corresponds to the length of oneACE domain. The neu-
All the Asn residues determined by Yu et al. [19] as
rolysin molecule structure determined by X-ray analy- subjected to glycosylation during the expression of tes-
sis predominantly consists of α-helices (53%) and ticular human ACE in the Chinese hamster ovary cells
unordered structure (41%) [21]. It also includes one (Asn666, Asn685, Asn731, Asn913, and Asn1162
β-sheet comprising approximately 6% of the amino according the numeration in this work) are close to the
acid residues present in the protein.
surface of the ACE C-domain in our model (Fig. 4).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

4
30
VORONOV et al.
Asn 666
NH2
Asn 685
Asn 644
His1089
His963
Glu960
Asn 913
Glu987
Asn 1162
His959
Asn 731
HOOC
Tyr751
Phe869
Tyr863
Tyr835
Possible localization
of the hydrophobic area
Fig. 4. The model of spatial folding of the polypeptide chain of the C-domain of ACE. The amino acid residues of the active site,
the sites of potential glycosylation, and the amino acid residues of the proposed hydrophobic area are marked.
This fact additionally confirms the correctness of our the mutant enzyme toward the thermal inactivation: the
model.
stability was higher than that of the C-domain but still
lower than that of the recombinant N-domain.
We failed to model with sufficiently high precision
the N-terminal structures of the 1–105 sequence of the
N-domain and of the 604–705 sequence of the C-
domain, which we regarded as most probably responsi-
ble for different thermal stabilities of the domains. In
Figures 4 and 5, one can see an incoherent protein
structure with many chain breaks in these regions,
which points to a low structural homology in these
regions. According to the modeling results, these
regions should occupy separate spatial positions. These
regions are rather distant from the active sites and form
structurally separate subdomains, which are probably
important for the general domain stability.
Despite the limitations of the proposed model, it
helped us find the single hydrophobic site on the sur-
face of the ACE C-domain that might bind the ANS
molecule and serve as a target for the damaging action
of the products of water radiolysis. This hydrophobic
cluster is formed by three Tyr residues (Tyr751, Tyr835,
and Tyr 863) and by one Phe residue (Phe869) (Fig. 4).
This hydrophobic site was rather distant from the active
site cleft, which well agrees with the experimentally
found fact that the ANS binding to the hydrophobic site
of ACE does not affect its catalytic activity [17].
The results of Marcic et al. [4] may confirm this
These Tyr and Phe residues are conserved and also
conclusion: the authors prepared a chimerical human present in the ACE N-domain where they, in principle,
ACE with the N-terminal region of C-domain (the 601– also can form a hydrophobic cluster. The fine arrange-
7
47 sequence) replaced by the corresponding region of ment of this cluster probably differs from that in the C-
N-domain (the 1–148 sequence). This replacement domain, and this difference could result in a signifi-
affected both the catalytic properties and the stability of cantly decreased binding of the dye molecule and fixing
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STRUCTURAL AND FUNCTIONAL PECULIARITIES
431
Asn82
Asn25
Asn117
Asn9
Asn45
Asn480
Asn289
Asn131
Fig. 5. The model of the spatial folding of the polypeptide chain of the N-domain of ACE. The amino acid residues of the active site
and the potential glycosylation sites are marked. The probable carbohydrate-binding site is indicated by a circle. This projection
was made from that given in Fig. 4 by a 90°-turn in the indicated direction relative to the given axis.
only one hydrophobic site on the globule of somatic cule [23]. The epitope recognized by the 9B9 antibody
enzyme under the experimental conditions.
on the surface of the somatic human ACE is situated
between Lys535 and Lys572 of the ACE N-domain
ACE is an unusual protein, because it contains a spe-
cific carbohydrate-binding site, which imparts lectin-
like properties to the enzyme [23]. The functions of this
site are not associated with the direct catalytic proper-
[
24]. These results help localize the carbohydrate-bind-
ing site in the enzyme N-domain.
This region was not included in our model of the
ties of ACE, but are probably related to the structural three-dimensional structure of N-domain. However, we
arrangement of the enzyme in biological membrane.An presume by analogy with the homologous region of C-
analysis of the incomplete model of the enzyme N- domain that this region should be situated in the space
domain allowed us to localize the possible region of region marked by a circle in Fig. 5. It is important that
this carbohydrate-binding site on the protein surface. It the majority of the sites of potential glycosylation are
was established (O.A. Kost and S.M. Danilov, unpub- situated on the same side of the ACE globule where we
lished results) that the 9B9 monoclonal antibody to the localized carbohydrate-binding site. Such mutual
N-domain of human ACE and its Fab-fragments shield arrangement of the glycosylation sites and carbohy-
the carbohydrate-binding site on the ACE globule and drate-binding site should create sterical hindrances for
inhibit the enzyme dimerization caused by the interac- the carbohydrate-controlled interaction of more than
tion of carbohydrate chains of one enzyme molecule two molecules simultaneously. Actually, only the for-
with the carbohydrate-binding site of the another mole- mation of ACE dimers was shown to be controlled by
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4
32
VORONOV et al.
carbohydrates, whereas larger aggregates of the strate concentrations varied in the range from 0.5 to 3 ×
enzyme result from other interactions [25].3
K . The enzyme concentration in the reaction mixture
m
was made to be equal to K determined in preliminary
i
experiments. The inhibitor concentration was varied
EXPERIMENTAL
within the range of 0.5–10 × [E] . The experimental
0
α
Materials. N -3-(2-Furyl)acryloyl-L-phenylalanyl- data were processed by the Henderson method [29].
glycyl-glycine,
lysyl-L-proline (lisinopril), (2S)-1-(3-mercapto-2-
methylpropionyl)-L-proline (captopril), and (S)-1-[N-
N-(S-1-carboxy-3-phenylpropyl)-L-
The ACE thermal inactivation was monitored by
measuring the residual activity of the enzyme (with the
Cbz-Phe-His-Leu substrate) after the incubation at the
preset temperature for various periods of time [5]. The
residual activity was calculated as percentage of the
activity of the intact enzyme.
(
1-[ethyloxycarbonyl]-3-phenylpropyl)-L-alanyl]-L-pro-
line maleate (enalapril maleate) were from Sigma
United States); hippuryl-L-histidyl-L-leucine and his-
(
α
tidyl-L-leucine were from Serva (Germany); and N -
benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine
was from Bachem (United States). N -3-(2-furyl)acryl-
oyl-L-phenylalanyl-L-alanyl-L-alanine and N -3-(2-
furyl)acryloyl-L-phenylalanyl-L-alanyl-L-lysine were
kindly gifted by M.V. Ovchinnikov (the Cardiological
Scientific Center of the Russian Academy of Medical
Effect of ANS on the kinetics of the radiation inac-
α
tivation of ACE in the solution was studied by exposure
–8
α
of a 10 M solution of the enzyme to γ-irradiation [17].
The value of absorbed radiation dose was determined
by the exposure time (the kinetical absorption of the
dose at a constant dose rate of 0.05 Gy/s). The residual
activity of the preparations was measured according to
the hydrolysis rate of the Cbz-Phe-His-Leu as a sub-
strate.
α
Sciences). (RS)-N -[1-Carboxy-2-(benzylaminocar-
α
bonyl)ethyl]-L-alanyl-L-proline (PP-09), and N -3-(2-
furyl)acryloyl-L-phenylalanyl-L-phenylalanyl-L-argin-
ine were a kind gift of V.F. Pozdnev (the Institute of
Prediction of the ACE secondary structure was
Biomedical Chemistry of the Russian Academy of performed using the Consensus service available on the
Medical Sciences).
Network Protein Sequence Analysis Web-server
France) http://npsa-pbil.ibcp.fr/. The structure was
(
Somatic and testicular ACE were isolated by the
method involving the enzyme extraction from bovine
lungs and testes with 50 mM phosphate buffer (pH 7.5)
containing 150 mM NaCl and 1 µM ZnCl in the pres-
ence of Triton X-100 and sequential cascade affinity
chromatography [26].
predicted simultaneously by eight various algorithms in
which the homology with the proteins of the known
crystal structures, the dependence of the conformation
of an amino acid residue on its nearest environment,
and the ability of an amino acid residue for the forma-
tion of hydrogen bonds were taken into account. The
most probable structural conformation of every region
was chosen on the basis of the results of all the predic-
tion algorithms. The accuracy of such prediction was
more than 70% according to [30].
2
N-Domain of ACE was prepared by a limited pro-
teolysis of the partially denatured somatic form of ACE
[5, 6].
All the preparations were homogenous according to
the SDS-PAGE. The concentrations of the active ACE
molecules were determined by the method of stoichio-
metric titration with lisinopril, a specific competitive
inhibitor of the enzymatic hydrolysis, with FA-Phe-
Gly-Gly substrate as described in [6].
Kinetics of the catalytic hydrolysis of FA-Phe-
Gly-Gly, FA-Phe-Ala-Ala, FA-Phe-Ala-Lys, and FA-
Phe-Phe-Arg were studied on a Simadzu UV-265FW
spectrophotometer (Japan) by the technique [27]. The
rates of catalytic hydrolysis of Hip-His-Leu and Cbz-
Phe-His-Leu were measured fluorimetrically on a Hita-
chi MPF-4 spectrofluorimeter (Japan) by measuring the
content of reaction product, His-Leu, with the use of o-
phthalic aldehyde [28].
Prediction of the motif of the spatial folding of
the peptide chain of the ACE domains was carried out
on the basis of the primary structures of the domains of
bovine ACE using the 3D-PSSM service [31] available
on the Web-server of the structural bioinformatics
group of the State College of Science, Technology and
Medicine (Great Britain), http://www.sbg.bio.ic.
ac.uk./servers/3dpssm/.
ACKNOWLEDGMENTS
This study was partially supported by the Russian
Foundation for Basic Research, project no. 00-04-482430.
Determination of the inhibition constants of the
single-domain forms of ACE by various competitive
inhibitors was performed under the equilibrium condi-
tions at a constant enzyme concentration and five sub-
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