Combined use of selective inhibitors and fluorogenic
substrates to study the specificity of somatic wild-type
angiotensin-converting enzyme
Nicolas D. Jullien
1
, Philippe Cuniasse
1
, Dimitris Georgiadis
2
, Athanasios Yiotakis
2
and Vincent Dive
1
1 CEA, De
´
partement d’Inge
´
nerie et d’Etudes des Prote
´
ines, Gif ⁄ Yvette, France
2 Department of Chemistry, Laboratory of Organic Chemistry, University of Athens, Greece
Angiotensin-converting enzyme (ACE) in vertebrates
is a zinc metallopeptidase involved in the release of
angiotensin II and the inactivation of bradykinin, two
peptide hormones that play a key role in blood pres-
sure regulation and renal and cardiovascular function
[1–4]. ACE inhibitors have been on the market for
more than 20 years, with successful applications for
conditions ranging from mild hypertension to post-
myocardial infarction [5,6]. Somatic ACE is a very
unusual enzyme which contains two active sites on the

d’Etudes des Prote
´
ines, 91191 Gif ⁄ Yvette
Cedex, France
Fax: +33 169089071
Tel: +33 169083585
E-mail:
(Received 19 December 2005, revised 16
February 2006, accepted 21 February 2006)
doi:10.1111/j.1742-4658.2006.05196.x
Somatic angiotensin-converting enzyme (ACE) contains two homologous
domains, each bearing a functional active site. Studies on the selectivity of
these ACE domains towards either substrates or inhibitors have mostly
relied on the use of mutants or isolated domains of ACE. To determine
directly the selectivity properties of each ACE domain, working with wild-
type enzyme, we developed an approach based on the combined use of
N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic
substrates. With this approach, marked differences in substrate selectivity
were revealed between rat, mouse and human somatic ACE. In particular,
the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a
strict N-domain-selective substrate of mouse ACE, whereas with rat ACE
it displayed marked C-domain selectivity. Similar differences in selectivity
between these ACE species were also observed with a new fluorogenic sub-
strate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of
these results, changes in amino-acid composition in the binding site of these
three ACE species were pinpointed. Together these data demonstrate that
the substrate selectivity of the N-domain and C-domain depends on the
ACE species. These results raise concerns about the interpretation of func-
tional studies performed in animals using N-domain and C-domain sub-
strate selectivity data derived only from human ACE.

discussed in previous papers [16,19], each part reflects
first the binding of RXP407 to the N-domain at low
inhibitor concentration, followed at higher concentra-
tions by inhibitor binding to the C-domain. The loca-
tion of the inflection points in this profile provides a
direct measure of the IC
50
value (concentration that
causes 50% inhibition) for both domains. In addition,
the value of the inhibition percentage, determined after
inhibitor binding to the active site showing the highest
affinity, was demonstrated to depend on the selectivity
with which the substrate is processed by the N-domain
and C-domain (indicated by the arrow in Fig. 1A).
Thus, information on the selectivity of both the sub-
strate and the inhibitor for the N-domain and
C-domain can be determined from such profiles.
Remarkably, with the same substrate, RXP407 inhi-
bition profiles of somatic ACE purified from mouse
(Fig. 1A, open circle) and rats (closed square) are quite
different from that observed for human ACE. For
mouse ACE, the shape of the inhibition profile sug-
gests the titration of a single active site to which
RXP407 binds with high affinity. Such a profile can be
expected for a substrate that is mostly cleaved by only
one active site, probably the N-domain given the
RXP407 selectivity. In contrast with mouse and human
ACE forms, titration of rat ACE by RXP407 yields a
profile that is shifted to the right. The shape of this
profile is consistent with the binding of RXP407 to the

P
O
-
H
N
CH
2
H
N
O
CH
3
NH
2
O
O
N
H
COO
-
O
H
3
C
O
H
3
C
RXP407
P

similar proposal, the higher catalytic efficiency of the
C-domain in cleaving the Mca-Ala substrate.
Titration of the mouse ACE C-domain using low
concentrations of RXPA380 (Fig. 1B, open circle) did
not affect enzyme activity. Inhibition of enzyme activ-
ity only occurred when RXPA380 started to bind to
the N-domain of ACE, at very high concentration.
This observation is in agreement with the RXP407
inhibition profile of mouse ACE, suggesting that the
Mca-Ala substrate is mostly cleaved by the N-domain
of mouse ACE.
To conclude this part, the inhibition profiles
obtained with both RXP inhibitors confirm that the
catalytic efficiency by which Mca-Ala is processed by
the N-domain and C-domain varies according to the
ACE species. For rat and human enzymes, estimation
of IC
50
values for the binding of the inhibitor to the
N-domain and C-domain can be deduced from the
inhibition profiles established with the Mca-Ala sub-
strate. For mouse ACE, as this substrate is mostly
cleaved by the N-domain, only IC
50
values for inhib-
itor binding to the N-domain can be obtained from
the inhibition profile. Thus, affinity of the inhibitor for
the mouse ACE C-domain cannot be determined using
this substrate. This limitation has been overcome by
developing another fluorogenic substrate of ACE.

displays C-domain selectivity, the degree of
which depends on the ACE species. Titration of the
ACE C-domain with RXPA380 promoted higher ACE
inhibition (55% to 90% inhibition, depending on the
ACE species, Fig. 1D) than blockade of the N-domain
by RXP407 (10% to 45% inhibition, depending on the
ACE species, Fig. 1C). Comparison of the profiles in
Fig. 1C,D shows that Mca-BK
(1)8)
displays the highest
C-domain selectivity toward rat ACE. In contrast with
Mca-Ala, IC
50
values for the binding of the RXP com-
pounds to both the N-domain and C-domain of mouse
ACE can be estimated using the Mca-BK
(1)8)
sub-
strate. From the similarities between the different
profiles, it can be concluded that the affinity and selec-
tivity of both inhibitors for the N-domain and
C-domain of different ACE species are conserved,
except for RXP407 affinity for the N-domain of rat
ACE (see below).
Catalytic efficiencies of the ACE N-domain and
C-domain in cleaving the Mca-Ala and
Mca-BK
(1)8)
substrates
The inhibition profiles described above indicate the

strate turns out to be an N-domain-selective substrate
of mouse ACE, showing the highest catalytic efficiency
in cleaving this substrate, as compared with human
and rat N-domain. The two active sites of human
ACE hydrolyze this substrate, but the N-domain is
more efficient than the C-domain in catalyzing this
reaction. The reverse is observed for rat ACE, this
substrate being better cleaved by the C-domain. Inter-
estingly, although significant differences between the
catalytic efficiency of either the N-domain or
C-domain are observed between these ACE species,
the overall activity of both domains is constant for the
three species. Thus, in this case, the gene duplication
of ACE may be a way to keep the catalytic efficiency
of the somatic enzyme intact, while allowing variations
in the N-domain and C-domain catalytic properties.
Overall, these data on interspecies Mca-Ala substrate
Table 1. Kinetic parameters for the hydrolysis of the Mca-Ala substrate by rat, mouse and human somatic ACE. Kinetic parameters k
cat
and
K
m
were obtained by inhibiting the activity of the C-domain of ACE with RXPA380 (active N-domain) or by inhibiting the N-domain activity
with RXP407 (active C-domain)(see Experimental procedures). N + C is the sum of the k
cat
⁄ K
m
values reported for the N-domain and
C-domain. k
cat

(s
)1
) 19.1 (17.9–20.0) ND 4.2 (37.–4.7)
K
m
(lM) 49.5 (43.1–53.3) ND 64.6 (52.4–77.1)
k
cat
⁄ K
m
(s
)1
ÆlM) 0.39 ND 0.06
N+C k
cat
⁄ K
m
(s
)1
ÆlM) 0.45 – 0.45
Somatic ACE k
cat
(s
)1
) 24.7 (22.5–26.3) 14.2 (13.6–15.8) 16.5 (15.1–17.7)
K
m
(lM) 46.9 (38.4–52.5) 24.6 (17.9–30.8) 34.8 (27.2–40.9)
k
cat

determine the K
i
values of the RXP compounds for
the N-domain and C-domain of each ACE species.
Assuming that each ACE active site functions inde-
pendently, we previously showed that simulated inhibi-
tion profiles that best reproduce the experimental data
can provide access to K
i
values [16,19]. Such simula-
tions rely on the use of inputs, notably the catalytic
parameters displayed by each ACE domain in cleaving
the substrate used in the experiments. The results
reported above, in the presence of RXP inhibitor
blocking only one active site, provide approximate val-
ues of the catalytic parameters displayed by each ACE
domain for cleaving the Mca-Ala and Mca-BK
(1)8)
substrates. These values were thus tentatively used to
simulate inhibition profiles able to reproduce the
experimental inhibition profiles. As shown in Fig. 1,
excellent fits between experimental data and simula-
ted curves were generally observed. The K
i
values as
determined by this approach (Table 3) indicate that
RXP407 and RXPA380 are, respectively, highly
N-domain-selective and C-domain-selective inhibitors
of the three ACE species, whatever the substrate used.
For human ACE, the K

cat
⁄ K
m
values
were calculated using the experimental k
cat
and K
m
values.
Parameter Rat Mouse Human
N-domain k
cat
(s
)1
) 38.4 (28.9–58.6) 8.9 (7.9–10.5) 17.8 (16.6–20.3)
K
m
(lM) 58.0 (22.9–65.6) 30 (18.8–36.4) 26.1 (18.9–31.3)
k
cat
⁄ K
m
(s
)1
ÆlM) 0.66 0.30 0.68
C-domain k
cat
(s
)1
) 110.9 (105.6–119.3) 4.1 (3.7–4.5) 15.7 (15.1–16.0)

)1
ÆlM) 5.89 0.59 2.79
Table 3. Potency and selectivity of RXP407 and RXPA380 toward
the N-domain and C-domain of human, mouse and rat somatic
ACE. K
i(app)
values were determined from the simulations that best
reproduced the inhibition profiles reported in Fig. 1. ND, not deter-
mined, no detectable activity.
Domain
K
i(app)
values (nM)
Mca-Ala Mca-BK
(1)8)
Rat Mouse Human Rat Mouse Human
RXP 407 N 55 8 10 30 13 16
C 8500 ND 4000 10000 6000 8000
RXP A380 N 7000 3500 5500 10000 5500 5500
C 2.6 ND 5 7 8 10
ACE substrate specificity N. D. Jullien et al.
1776 FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS
RXPA380 in complex with human ACE [20]. In partic-
ular, two bulky and hydrophobic residues of the human
ACE C-domain, Val955 and Val956 located in the S
2
¢
subsite, were proposed to provide favorable interactions
with the tryptophan side chain of RXPA380. These
interactions should be lost in the N-domain, as valine

¢ and S
2
¢ pockets, two residues
may greatly influence the RXP407 potency and selectiv-
ity. In fact, in the N-domain, the aspartyl side chain
of RXP407 is observed to interact with Tyr369 and
Arg381 through hydrogen-bond contacts. Indeed, short
distances between the O
d1
and O
d2
atoms of the
RXP407 aspartyl residue and Tyr369 ⁄ O
g
atom on one
side and Arg381 ⁄ N
f
,N
g
atoms on the other side are
observed in this model (Fig. 2). Similar interactions
cannot take place in the C-domain, as these two resi-
dues are replaced by Phe967 and Glu979. As the aspar-
tyl residue in RXP407 is the key residue controlling
inhibitor selectivity, we suggest that the mutations
observed in the 369 ⁄ 967 and 381 ⁄ 979 positions may
contribute to RXP407 selectivity. Rat and mouse ACE
display the same feature as is observed in human ACE,
which is consistent with the potency and selectivity dis-
played by RXP407 toward the rat and mouse enzymes.

2
¢ Ser260 Ser260 Ser260 Thr858 Thr858 Thr858
Asp354 Glu354 Asp354 Glu952 Glu952 Glu952
Ser357 Ala357 Ser357 Val955 Val955 Val955
Thr358 Thr358 Thr358 Ile956 Ile956 Val956
Glu431 Glu431 Glu431 Asp1029 Asp1029 Asp1029
Fig. 2. Detail of the human ACE N-domain active in the interaction
with RXP407. The active-site helix carrying the HEXXH sequence is
colored yellow. RXP407 is colored by atom type; the carbon atoms
are in green. Active-site residues located at a distance less than
5A
˚
from RXP407 are in light blue when they are observed to
change between ACE species (in either the N-domain or C-domain).
Three residues interacting with RXP407 in the model are displayed
in orange; these residues are conserved between the N-domain of
the three ACE species (N-domain numbering). Distances in ang-
stroms between atoms of Arg381, Tyr369 and the aspartate atoms
of RXP407 are reported. Hydrogens and zinc atom are omitted. The
figure was prepared with
PYMOL software.
N. D. Jullien et al. ACE substrate specificity
FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS 1777
Tyr369 and Arg381 residues were observed to interact
with the acetyl group of RXP407. As mentioned above,
our model seems to better explain the key role of the
aspartyl residue in inhibitor selectivity. As shown in
Fig. 2 (see also Table 4), the model of RXP407 interac-
tion with the N-domain reveals that several positions in
the ACE active site, covering the S

approach, the catalytic property of each domain
towards the hydrolysis of a substrate can be determined
without the need to produce mutants or isolate ACE
domains. The results of this approach are also consis-
tent with the shape of the inhibition profiles observed
for each ACE species. Intuitive interpretation of the
inhibition profiles of mouse and rat somatic ACE with
RXP407 and RXPA380 (Fig. 1A,B) suggests that the
Mca-Ala substrate is a strict N-domain-selective sub-
strate of mouse ACE, whereas it is almost cleaved by
the C-domain of rat ACE. This interpretation is con-
firmed by the kinetic parameters reported in Table 1.
Overall, this work based on the study of two syn-
thetic substrates and three different ACE species
reveals that the N-domain and C-domain substrate
selectivity is not conserved between different ACE spe-
cies, implying that the functional role played by each
domain may change from one species to another. Thus,
any conclusion on the N-domain and C-domain sub-
strate selectivity based on a single ACE species could
be misleading. In this respect, it is worth mentioning
that the N-domain and C-domain selectivity towards
physiological substrates of ACE (angiotensin I, brady-
kinin and N-acetyl-seryl-aspartyl-lysyl-proline) has been
established only for the human enzyme [21], because of
the availability of a mutant form of the human enzyme
containing a single functional active site. As far as sub-
strate selectivity is concerned, data obtained in animal
models could be misinterpreted if the properties of the
human enzyme are used. The approach presented in

domain-selective ACE inhibitors may vary between rat
and mouse models, rendering extrapolation of these
results to the human situation problematic.
In support of our experimental results highlighting
differences in N-domain and C-domain substrate selec-
tivity between ACE species, our nonexhaustive compar-
ison of mouse, rat and human ACE sequences around
the active site of these enzymes identifies several resi-
dues that change (Table 4). In human ACE, it has
already been reported that Asn494 occurs in an N-gly-
cosylation sequon (NTV), that is unique to the N-
domain [6]. Any glycosylation of this residue, which is
located in the active site, is expected to greatly influence
enzyme activity. Interestingly, this asparagine is
replaced by serine in the rat N-domain. Whether this
mutation results in no glycosylation or O-glycosylation
of Ser494 in rat ACE is not known [22], but, in any
case, it can be expected to affect enzyme activity.
The low efficiency of the rat N-domain in cleaving the
ACE substrate specificity N. D. Jullien et al.
1778 FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS
Mca-Ala substrate, compared with the N-domain of
the mouse and human enzymes, may in part be due to
this mutation. The resolution of the 3D structure of the
germinal form of ACE has provided a strong impetus
for further studies aimed at understanding, at the
molecular level, how the two active sites of somatic
ACE function [18]. The data reported in this study pro-
vide supplementary information about the residues that
should be considered in any ACE models intended to

dicale, Unite
´
36,
Paris, France. Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH
(Mca-BK
(1)8)
) substrate [Mca, (7-methoxycoumarin-4-
yl)acetyl; DpaOH, N
3
-(2,4-dinitrophenyl)-l-2,3-diaminopro-
pionyl)] was prepared by following the procedure described
for Mca-Ala-Ser-Asp-Lys-DpaOH (Mca-Ala) [16].
Enzymes
Human wild-type somatic ACE was obtained by stable
expression in Chinese hamster ovary cells transfected with
appropriate ACE cDNA [14]. This material was kindly pro-
vided by P. Corvol (Colle
`
ge de France, Paris, France).
Expression and purification of ACE were performed as pre-
viously described [14]. Mouse and rat somatic ACE were
purified by affinity chromatography as described previously
[17], from lung homogenates obtained from C57BL ⁄ 6 mice
and Lewis rats (Charles River France, L’arbresle, France).
ACEs purified by this method appeared as homogeneous
single bands on SDS ⁄ PAGE.
Enzyme assays
Continuous assays were performed by recording the fluores-
cence increase at 405 nm (e
ex

and k
cat
for the hydro-
lysis of the substrates Mca-Ala and Mca-BK
(1)8)
by the
N-domain or the C-domain of human, mouse, or rat ACE
were determined by blocking the activity of the C-domain
by 150 nm RXPA380 or the activity of the N-domain by
350 nm RXP407, except for rat ACE for which the
N-domain was inhibited using 750 nm RXP407. At these
concentrations, RXP compounds mainly inhibit the activity
of one active site, allowing the determination of the kinetic
parameters of the other active site, free of inhibitor. For
each species and each inhibitor, ACE was incubated with
the inhibitor for 45 min before substrate addition. The kin-
etic parameters were determined using the direct linear plot
method [27–30] and substrate concentration ranges of 10–
122 lm for Mca-Ala and 2–71 lm for Mca-BK
1-8
. Concen-
trations of ACE in these experiments were determined by
titration of the enzyme with quinaprilat.
Inhibition studies
For inhibition studies, all inhibitors were preincubated for
45 min before initiation of the reaction by substrate addition.
The substrate concentration used was 8 lm for Mca-Ala and
5 lm for Mca-BK
(1)8)
. Data were collected every 30 s over a

⁄ K
m
)
C
ratio constant.
Identification of the residues covering the
S
2,
S
1,
S
1
¢ and S
2
¢ binding sites in ACE
A model of RXP407 interaction with the human ACE N-
domain has been developed to identify residues of the S
2
,
S
1
,S
1
¢ and S
2
¢ pockets that could influence either inhibitor
or substrate selectivity in this enzyme. The conservation of
these residues was then checked, using aligned sequences of
the N-domain and C-domain of human, mouse and rat
enzymes. The model of the N-domain of human ACE

ÆA
˚
)2
for the ions and their chelating residues, the
atoms of the protein situated at a distance greater than 5 A
˚
of the inhibitor, and the backbone and Cb atoms of the
inhibitor and those of the protein located at a distance
smaller than 5 A
˚
of the inhibitor, respectively. No har-
monic restraints were applied to hydrogen atoms. The ini-
tial step of the relaxation protocol consists of an initial
2000 cycles of Adopted Basis Newton-Raphson energy min-
imization. Then 100 000 steps of molecular dynamics using
the Verlet algorithm were undertaken. The integration step
was set to 0.0004 ps. The temperature was gradually
increased by 25 K each 1000 steps to reach 300 K. This
molecular dynamics was followed by 5000 cycles of energy
minimization. During the calculations, the nonbonded
interactions were modeled using a Lennard-Jones function
and a coulombic electrostatic term with a nonbonded cut-
off of 16 A
˚
. The dielectric constant was set to 1. The result-
ing structure was then analyzed with the program pymol
(Delano Scientific Inc., San Francisco, CA, USA).
Acknowledgements
This work was supported by the CEA ( Commissariat a
`

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