Báo cáo khoa học: Structural diversity of angiotensin-converting enzyme Insights from structure–activity comparisons of two Drosophila enzymes docx

Structural diversity of angiotensin-converting enzyme
Insights from structure–activity comparisons of two Drosophila
enzymes
Richard J. Bingham
1
, Vincent Dive
2
, Simon E. V. Phillips
1
, Alan D. Shirras
3
and R. Elwyn Isaac
1
1 Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, UK
2 Departement d’Etudes et d’Ingenierie des Proteines, Commissariat a l’Energie Atomique, CE-Saclay, Gif-Sur-Yvette, France
3 Department of Biological Sciences, University of Lancaster, UK
Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is
a zinc peptidyl-dipeptidase, which is best known for
catalysing the last step in the synthesis of the vasocon-
strictor angiotensin II (AII) from angiotensin I (AI)
and for the metabolic inactivation of the vasodilator
bradykinin (BK) [1]. The somatic form of the enzyme
is a glycosylated type I membrane protein comprising
two homologous domains, generally known as the
N-domain and C-domain, arranged in tandem and
joined by a short connecting peptide sequence [2].
Keywords
ACE inhibitors; angiotensin-converting
enzyme (ACE); Drosophila melanogaster;
peptide metabolism; peptidyl-dipeptidase
Correspondence

R. E. Isaac, Faculty of Biological Sciences,
Miall Building, University of Leeds,
Leeds LS2 9JT, UK
Fax: +44 113 34 32835
Tel: +44 113 34 32903
E-mail:
(Received 21 September 2005, revised
15 November 2005, accepted 21 November
2005)
doi:10.1111/j.1742-4658.2005.05069.x
The crystal structure of a Drosophila angiotensin-converting enzyme
(ANCE) has recently been solved, revealing features important for the
binding of ACE inhibitors and allowing molecular comparisons with the
structure of human testicular angiotensin-converting enzyme (tACE).
ACER is a second Drosophila ACE that displays both common and dis-
tinctive properties. Here we report further functional differences between
ANCE and ACER and have constructed a homology model of ACER to
help explain these. The model predicts a lack of the Cl
–
-binding sites, and
therefore the strong activation of ACER activity towards enkephalinamide
peptides by NaCl suggests alternative sites for Cl
–
binding. There is a
marked difference in the electrostatic charge of the substrate channel
between ANCE and ACER, which may explain why the electropositive
peptide, MKRSRGPSPRR, is cleaved efficiently by ANCE with a low K
m
,
but does not bind to ACER. Bradykinin (BK) peptides are excellent ANCE

substrates. Models of BK docked in the substrate channel suggest that the
peptide adopts an N-terminal b-turn, permitting a tight fit of the peptide in
the substrate channel. This, together with ionic interactions between the
guanidino group of Arg9 of BK and the side chains of Asp360 and Glu150
in the S
2
¢ pocket, are possible reasons for the high-affinity binding of BK.
The replacement of Asp360 with a histidine in ACER would explain the
higher K
m
recorded for the hydrolysis of BK peptides by this enzyme.
Other differences in the S
2
¢ site of ANCE and ACER also explain the selec-
tivity of RXPA380, a selective inhibitor of human C-domain ACE, which
also preferentially inhibits ACER. These structural and enzymatic studies
provide insight into the molecular basis for the distinctive enzymatic fea-
tures of ANCE and ACER.
Abbreviations
ACE, angiotensin-converting enzyme; ANCE, Drosophila melanogaster angiotensin-converting enzyme; ACER, Drosophila melanogaster
angiotensin-converting enzyme-related; BK, bradykinin; AI, angiotensin I; AII, angiotensin II; Abz, o-aminobenzoic acid; Hip-His-Leu, hippuryl-
L-histidyl-L-leucine.
362 FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS
Each domain is catalytically active, and both are cap-
able of cleaving AI and BK. The ACE gene also gives
rise to a second mammalian ACE, known as either tes-
tis (tACE) or germinal ACE, through the use of an in-
tragenic promoter that drives expression in developing
spermatocytes. It is a single-domain enzyme that is
identical with the C-domain of somatic ACE, apart

from a peptide insert encoded by the testis-specific
exon 13 of the ACE gene [2]. ACE knockout mice dis-
play renal abnormalities, low blood pressure, anaemia
and male infertility, confirming the important role of
this enzyme in development, blood homoeostasis and
reproduction [2].
Although N-domain and C-domain are highly similar
in protein sequence and share many enzymatic proper-
ties, they can be differentiated by substrate and inhib-
itor preferences and by the extent to which they are
activated by Cl
–
[3–5]. The haemoregulatory peptide,
N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), another in vivo
substrate for mammalian ACE, is hydrolysed more effi-
ciently by the N-domain, as is the internally quenched
fluorogenic substrate Abz-SDK(Dnp)P [6,7]. Cl
–
can
stimulate the activity of both ACE domains, but the
C-domain active site is more sensitive to changes in
Cl
–
concentration [3]. The level of activation, as well as
the concentration of Cl
–
required for maximal stimula-
tion, is dependent on pH and the peptide substrate. The
two domains of mammalian ACE can also be distin-
guished by the N-domain-selective inhibitor RXP407

[8], the C-domain-selective inhibitor RXPA380 [9], and
several BK-potentiating peptides [10].
A homologue of ACE, known as ACE2, has been
characterized as a single-domain type I glycoprotein
[11,12]. It is important for normal contractility of heart
muscle [13]. The important enzymatic feature of ACE2
is that, unlike ACE, it is a carboxypeptidase, removing
a single residue from the C-terminus of peptides that
have either a Pro or Leu in the P
1
position, e.g. angio-
tensin II, apelin 13 and dynorphin A 1–13 [14]. The
activity of ACE2 is greatly enhanced in the presence
of NaCl [15,16]. Therefore Cl
–
activation is a common
feature of the mammalian members of the ACE family
of peptidases.
In vertebrates, the number of ACE genes appears to
be limited to ACE and ACE2, but in some insects
there has been a much greater expansion of this gene
family. For example, in the mosquito, Anopheles
gambiae, and in Drosophila melanogaster there are nine
and six ACE genes, respectively [17,18]. Of the six
Drosophila genes, only ANCE and ACER have been
confirmed to produce functional metallopeptidases
[19,20]. They are both single-domain proteins with
 40% amino-acid sequence identity and 60% similar-
ity to each of the two domains of mammalian ACE.
ANCE and ACER have distinct tissue expression pat-

terns, indicating different physiological roles [21,22].
ANCE appears to have a role in embryogenesis, meta-
morphosis and reproduction [20,23,24]. A function for
ACER has not been established, but the protein is
associated with the developing heart in embryos
and in the brain and reproductive tissues of adults
(A. Carhan, R.E. Isaac and A.D. Shirras, unpublished
results). The two enzymes share some enzymatic prop-
erties, such as peptidyl-dipeptidase activity towards
hippuryl-l-histidyl-l-leucine (Hip-His-Leu), and BK,
and inhibition by inhibitors of mammalian ACE
[19,20,25]. However, compared with ANCE, ACER
displays more restricted substrate specificity. Although
both ANCE and ACER hydrolyse Hip-His-Leu, only
the ANCE activity is enhanced in the presence of
NaCl [20,25]. Another interesting difference between
ACER and ANCE is that the ACER active site, but
not that of ANCE, can accommodate an N-domain-
specific inhibitor (RXP407), indicating common active-
site features for ACER and the N-domain of human
ACE [17].
Recent descriptions of the high-resolution molecular
structure of ACE–inhibitor complexes for both human
tACE [26,27] and Drosophila ANCE [28] have revealed
the molecular details of the active site and how ACE
inhibitors bind with high affinity. These studies con-
firm many of the predictions regarding the identity of
the active-site residues and, in the case of tACE, iden-
tify other side chains involved in the binding of Cl
–

at
two sites (Cl1 and Cl2) positioned outside of the active
site. The crystal structure of human ACE2, with and
without bound inhibitor, has also recently been repor-
ted [29] and has provided a structural explanation for
why ACE2 is a carboxypeptidase and not a peptidyl-
dipeptidase. The structure of the native ACE2 identi-
fied a single Cl
–
-binding site that corresponded to the
Cl1 site of tACE. No bound Cl
–
was recognized in the
crystal structure of Drosophila ANCE, and it has been
proposed that the equivalent Cl
–
-binding sites in
ANCE are substantially different and, in the case of
Cl2, may be absent [26], which may explain the weaker
effect of Cl
–
on enzyme activity reported for this
enzyme. In ACE2, the Cl2 site also does not exist,
which leaves only Cl1 as a recognized Cl
–
-binding site
[29]. Interestingly, an alternative, but undefined, bind-
ing site for Cl
–
has been suggested, which may be influ-

ential in the conformational movement that occurs on
formation of the ACE2 ES complex [26,29].
Comparative molecular and biochemical studies of
members of the ACE family are likely to provide new
insights into the evolution of the ACE active site, the
R. J. Bingham et al. Structure-activity of Drosophila ACEs
FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS 363
structural basis for differences in substrate specificity
and the mechanisms by which Cl
–
can have profound
effects on enzyme activity. In this respect, Drosophila
ANCE and ACER appear to be good examples of two
family members that have diverged in structure and
substrate specificity and are therefore likely to provide
valuable information. We now report on additional
biochemical differences between ANCE and ACER
regarding substrate specificity, the effect of Cl
–
on
enzyme activity, and inhibition by new domain-select-
ive inhibitors of human ACE. A model of the structure
of ACER has been generated, which provides explana-
tions for some of these biochemical differences.
Results
Hydrolysis of AI
The effect of NaCl on the conversion of AI into AII by
ANCE was determined at two pH values. At pH 7,
increasing the concentration of NaCl resulted in a faster
rate of conversion, which reached a plateau at 150–

200 mm NaCl (Fig. 1A). At pH 8, maximal activity was
achieved in the absence of NaCl, which had a weak
inhibitory effect on the hydrolysis of AI as the salt con-
centration increased from 0 to 200 mm NaCl (Fig. 1A).
To further examine the effect of NaCl and pH on the
mechanism of ANCE activation, the kinetic constants
of AI hydrolysis were determined in the presence and
absence of 100 mm NaCl at pH 7 and 8 (Table 1). The
activation by NaCl at pH 7 was the result of a 330%
increase in k
cat
⁄ K
m
, which was solely attributable to a
lowering of the K
m
. A similar rise in the k
cat
⁄ K
m
was
observed when the pH was increased from 7 to 8 in the
absence of NaCl, but in this case the greater catalytic
efficiency was achieved by a combined increased k
cat
and a lower K
m
. Although AI is an extremely poor sub-
strate for ACER, it was possible to determine kinetic
constants for this reaction (K

m
1.58 ± 0.28 mm; k
cat
0.01 ± 0.001 s
)1
), which showed that this marked dif-
ference between ACER and ANCE was due to the very
low k
cat
for AI hydrolysis by ACER. This weak pept-
idyl-dipeptidase activity, unlike that of ANCE and
mammalian ACE, was not stimulated by NaCl
(Table 2).
Hydrolysis of enkephalin peptides
[Leu5]Enkephalin, [Met5]enkephalin and their respect-
ive C-terminal amidated forms are hydrolysed at the
Gly-Phe bond by both ANCE and ACER at neutral
pH [20]. The endopeptidase activity of ACER, but
not ANCE, towards [Leu5]enkephalinamide and
[Met5]enkephalinamide was stimulated in the presence
of Cl
–
ions (Table 2). The enhancement of the hydro-
lysis of the amidated peptides by 500 mm NaCl was
12-fold and 15-fold, respectively, whereas the cleavage
of both [Leu5]enkephalin and [Met5]enkephalin was
inhibited by  50% (Table 2). The NaCl-induced activ-
ity of ACER was measured at different [Leu5]enkeph-
alinamide and [Met5]enkephalinamide concentrations,
which generated anomalous kinetics, including sub-

strate inhibition at peptide concentrations above
150 lm (data not presented).
Fig. 1. (A) Effect of NaCl on the conversion of AI (200 lM) into AII
by ANCE. Enzyme activity was measured using HPLC to quantify
the formation of AII in Hepes buffer (h,pH7;n, pH 8) in the pres-
ence of NaCl (0–200 m
M) as described in Experimental procedures.
The enzyme activity is expressed as percentage of the maximum
activity recorded at pH 8 in the absence of NaCl. Values are the
mean of three assays and the percentage standard error of
the mean was 1–4%. (B) Inhibition of ANCE and ACER by
MKRSRGPSPRR. Enzyme activity was determined using Abz-
YRK(Dnp)P as described in Experimental procedures and is
expressed as a percentage of the uninhibited activity.
Structure-activity of Drosophila ACEs R. J. Bingham et al.
364 FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS
Hydrolysis of BK and related peptides
Initial velocities for the hydrolysis of the BK peptides
were obtained by determining the rate of release of the
C-terminal dipeptide (Phe-Arg for BK, [Thr6]BK and
Ile-Ser-BK; Tyr-Arg for [Tyr8]BK). ANCE consis-
tently cleaved these peptides with much greater effi-
ciency (k
cat
⁄ K
m
) than ACER, mainly because of the
lower affinity of ACER for these substrates (Table 3).
In the case of ANCE, extending BK at the N-terminus
with Ile-Ser had no significant effect on the K

m
and
k
cat
, and replacing the Phe8 of BK with tyrosine resul-
ted in a modest increase in both the K
m
and k
cat
.In
contrast, replacing Ser6 of BK with threonine resulted
in greatly increased affinity between the substrate and
ANCE, but not ACER. Indeed the K
m
value for the
hydrolysis of [Thr6]BK was so low that it was difficult
to obtain accurate K
m
values using HPLC to quantify
reaction rates at very low substrate concentrations. We
therefore used [Thr6]BK as an inhibitor of the hydro-
lysis of Abz-YRK(Dnp)P and obtained a K
i
value of
23 ± 4 nm, confirming the very high affinity displayed
by ANCE for this peptide.
MKRSRGPSPRR is an invertebrate BK-like peptide
predicted to be a cleavage product of a neuropeptide
precursor gene in Aplysia californica [30]. HPLC analy-
sis showed that MKRSRGPSPRR was an excellent

substrate for ANCE, but was resistant to hydrolysis
by ACER. MS confirmed that reaction products
were MKRSRGPSP ([M +H]
+
, m ⁄ z 1014.3) and
MKRSRGP ([M +H]
+
, m ⁄ z 830.4), generated by
the sequential cleavage of Arg-Arg and Ser-Pro.
MKRSRGPSPRR was a strong inhibitor of the hydro-
lysis of Abz-YRK(Dnp)P with a K
i
of 185 nm for
the inhibition of ANCE (Fig. 1B). In contrast,
MKRSRGPSPRR, even at a concentration of 100 lm,
did not significantly inhibit ACER activity, measured
with the same fluorogenic substrate.
Homology model of the structure of ACER
We generated a model of ACER based on the crys-
tal structure of ANCE. The homology model
Table 1. Effect of NaCl on the kinetic constants for the conversion of AI into AII by ANCE. Kinetic constants for the conversion of AI into AII
were determined as described in Experimental procedures and are expressed as the mean ± SEM (n ¼ 3).
0m
M [NaCl] 100 mM [NaCl]
K
m
(mM) k
cat
(s
)1

) k
cat
⁄ K
m
(s
)1
ÆlM
)1)
K
m
(mM) k
cat
(s
)1
) k
cat
⁄ K
m
(s
)1
ÆM
)1
)
pH 7.0 2.70 ± 0.67 6.84 ± 1.04 2.53 · 10
)3
0.82 ± 0.14 6.83 ± 0.46 8.33 · 10
)3
pH 8.0 1.23 ± 0.17 11.06 ± 0.86 8.99 · 10
)3
1.04 ± 0.21 10.78 ± 1.12 10.37 · 10

)3
Table 2. Effect of NaCl on the hydrolysis of peptides by ACER. The
rate of hydrolysis of peptides (200 l
M) was determined in 0.1 M
Hepes ⁄ 10 lM ZnSO
4
, pH 7 as described in Experimental proce-
dures. Values are mean ± SEM (n ¼ 3).
Substrate
Reaction rate (units ⁄ h)
0m
M NaCl 500 mM NaCl
AI
a
0.033 ± 0.002 0.025 ± 0.002
[Leu5]Enkephalin
b
36.9 ± 1.0 19.6 ± 1.6
[Met5]Enkephalin
b
23.8 ± 0.03 9.9 ± 1.0
[Leu5]Enkephalinamide
b
10.1 ± 0.4 123.2 ± 1.8
[Met5]Enkephalinamide
b
3.3 ± 0.1 49.3 ± 4.0
a
Units of activity, nmol AII formed per lg ACER.
b

Units of activity,
nmol dipeptide released per lg ACER.
Table 3. Kinetic constants for the hydrolysis of bradykinin-related peptides by ANCE and ACER. –, No detectable hydrolysis of the peptide
and no inhibition of the cleavage of Abz-YRK(Dnp)P by ACER.
Substrate
ANCE ACER
K
m
(lM) k
cat
(s
)1
) k
cat
⁄ K
m
s
)1
(lM)
)1
K
m
(lM) k
cat
(s
)1
) k
cat
⁄ K
m

(s
)1
ÆlM
)1
)
BK (RPPGFSPFR) 0.27 ± 0.05 1.09 ± 0.03 4.04 4.88 ± 0.97 0.54 ± 0.03 0.09
Ile-Ser-BK (ISRPPGFSPFR) 0.30 ± 0.005 1.10 ± 0.005 3.66 5.54 ± 0.98 0.17 ± 0.01 0.03
[Tyr8]BK (RPPGFSPYR) 0.58 ± 0.08 2.41 ± 0.12 4.15 11.3 ± 3.46 1.17 ± 0.11 0.10
[Thr6]BK (RPPGFTPFR) 0.073 ± 0.04 0.24 ± 0.008 3.31 5.67 ± 1.58 0.60 ± 0.04 0.11
MKRSRGPSPRR 0.37
a
± 0.1 18.8
b
± 0.5 50.81 – – –
a
K
m
determined from the IC
50
value obtained by measuring initial rates of hydrolysis of the fluorogenic substrate Abz-YRK(Dnp)P (5 lM)in
the presence of different concentrations of MKRSRGPSPRR.
b
Estimated from the initial velocity recorded at a substrate concentration 100
times greater than the K
m
.
R. J. Bingham et al. Structure-activity of Drosophila ACEs
FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS 365
allowed us to compare the structure of the sub-
strate ⁄ inhibitor binding sites between these related

enzymes, which are very similar in primary protein
structure, but display quite different enzymatic prop-
erties. One of the striking differences between ANCE
and ACER predicted by our model is a significant
change in the electrostatic charge that lines the sub-
strate-binding channel, a change from predominantly
negative charges in ANCE to positive charges in
ACER (Fig. 2). To gain insight into why BK pep-
tides bind with higher affinity to ANCE than to
ACER, we docked BK and [Thr6]BK into the
ANCE substrate channel. The modelling predicts
that the negatively charged side chain of Asp360, as
well as Glu150, forms favourable ionic interactions
with the positively charged C-terminal arginine of
both substrates (Fig. 3). Interestingly, in ACER, this
interaction is lost because Asp360 is replaced with
His368 (Table 4). The models of BK and [Thr6]BK
bound to ANCE suggest that the extra methyl group
of [Thr6]BK occupies a small hydrophobic pocket,
which is conserved in both ANCE and ACER. The
models also suggest that the two peptides bind in a
similar orientation, with a b-turn centred on the resi-
dues Pro2-Pro3.
Selective inhibitors of ANCE and ACER
Inhibition constants were determined for RXPA380,
RXPA381 and RXPA384 for both ANCE and ACER
(Table 5). These values showed that RXPA384 was
only slightly more potent as an inhibitor of ACER,
Fig. 2. Surface representations of the elec-
trostatic potential of ANCE and a homology

model of ACER. The proteins have been
sliced in half to show the internal substrate-
binding channel. The N-chamber and
C-chamber (N and C) are postulated to bind
up to  7 N-terminal residues and the C-ter-
minal dipeptide of substrate, respectively.
Molecular surfaces and electrostatic poten-
tial were calculated with the program
SPOCK
(). ANCE co-ordi-
nates were obtained from the recently
determined crystal structure (PDB accession
code 1J36). The homology model of ACER
was generated in
SWISS-MODEL using the
ANCE structure as a template. Positive and
negative charges are represented by shades
of blue and red, respectively, with neutral
areas coloured white.
Fig. 3. A stick diagram showing predicted electrostatic interactions
between the C-terminal Arg9 of BK and ANCE. The interactions
between Asp360 of ANCE and the guanidino group of Arg9 of BK
will be lost in ACER as Asp360 is replaced with His368.
Structure-activity of Drosophila ACEs R. J. Bingham et al.
366 FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS
whereas RXPA381 was able to distinguish between the
two enzymes with a selectivity factor of more than 100
in favour of ACER. RXPA380 inhibited ACER with a
K
i

of 4.8 lm, but did not inhibit ANCE, even at a con-
centration of 100 lm.
To understand the molecular basis behind the select-
ive inhibition of ACER by RXPA380 and RXPA381,
these molecules were modelled into the binding sites of
ANCE and ACER. The model of RXPA380 ⁄ ACER
shows that RXPA380 is bound in a very similar orien-
tation to the model generated for RXPA380 ⁄ C-domain
ACE [31]. Phe1033 and Phe1103 of C-domain ACE
are important in forming a hydrophobic side of the S2¢
pocket for binding the tryptophan of RXPA380. Both
of these residues are conserved in ANCE, but in
ACER, Phe1103 is replaced with His519 (Table 4).
The other side of the S2¢ pocket is formed by two
adjacent valine residues in C-domain ACE (Val955
and Val956). Val955 is replaced by larger phenylalan-
ine and tyrosine residues in ANCE and ACER,
respectively, which in our models are pointing away
from the inhibitor so that the change in the size of the
side chain may have minimal effect on binding. Val956
of C-domain ACE is conserved in ACER as Val372,
but in ANCE this is replaced by Thr364, which redu-
ces the hydrophobicity of the ANCE S
1
¢ pocket
(Fig. 4A). In ANCE, Gln266 with its large polar side
chain replaces Ser275 and Thr858 of ACER and
C-domain ACE, respectively (Table 4). In our model,
the larger side chain of Gln266 restricts the space
available and results in steric hindrance of the large

indole ring of RXPA380 (Fig. 4A).
In RXPA381, the P
1
¢ and P
2
¢ proline and trypto-
phan residues of RXPA380 are replaced by smaller
alanine residues. The models of RXPA381 bound to
Table 4. Comparison of the residues that contribute to the S
2
¢ sub-
site of human C-domain ACE (the residue numbers for human tACE
are in parentheses) with the N-domain of human ACE, ANCE and
ACER.
N-domain
ACE
C-domain
ACE (tACE) ANCE ACER
Gln259 Gln857 (281) Gln265 Gln274
Ser260 Thr858 (282) Gln266 Ser275
Asp354 Glu952 (376) Asp360 His368
Ser357 Val955 (379) Phe363 Tyr371
Thr358 Val956 (380) Thr364 Val372
Asp393 Asp991 (415) Asp399 Asp407
Glu431 Asp1029 (453) Asp437 Ser445
Phe435 Phe1033 (457) Phe441 Phe449
Phe438 Phe1036 (460) Phe444 Phe452
Lys489 Lys1087 (511) Lys495 Lys503
Tyr498 Tyr1096 (520) Tyr504 Tyr512
Tyr501 Tyr1099 (523) Tyr507 Tyr515

Phe505 Phe1103 (527) Phe511 His519
Table 5. Potency of RXPA series of compounds as inhibitors of
ANCE and ACER. ANCE and ACER activities were measured using
the fluorogenic substrate Abz-YRK(Dnp)P (5 l
M) as described in
Experimental procedures. –, No inhibition with 100 l
M RXPA380.
Inhibitor
K
i
(nM)
ANCE ACER
RXPA380 (Cbz-Phew[PO
2
-CH]Pro-Trp-OH) – 4800
RXPA381 (Cbz-Phew[PO
2
-CH]Ala-Ala-OH) 365 3
RXPA384 (Cbz-Phew[PO
2
-CH]Ala-Trp-OH) 152 95
A
B
Fig. 4. Representations of enzyme–inhibitor interactions. (A)
RXPA380 bound to C-domain ACE (grey), superimposed on the
crystal structure of ANCE (yellow), highlighting differences between
the proteins at the S
2
¢ pocket. The absence of inhibition of ANCE
by RXPA380 can be explained by the replacement of Thr858 with

the larger Gln266, and Val956 with the polar Thr364. The combined
effects of these changes will be to reduce the hydrophobic nature
of the S2¢ site and restrict the space available for the large indole
ring of RXPA380. In ACER, the equivalent of Thr858 of C-domain
ACE is the smaller Ser275, whereas Val956 is conserved as
Val372. This is consistent with the inhibition of ACER by RXPA380.
(B) Space-filling representation of RXPA381 bound to ANCE (left)
and ACER (right) in the S
2
¢ pocket, comparing the differences in
packing of the P
2
¢ methyl group of RXPA381 (arrowhead) against
Val372 of ACER or the equivalent Thr364 in ANCE. The tyrosine
and lysine residues interacting with the C-terminus of the inhibitor
are labelled. The figure was generated in
PYMOL.
R. J. Bingham et al. Structure-activity of Drosophila ACEs
FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS 367
ANCE and ACER show that the inhibitor is bound in
a similar orientation, but with variation in the orienta-
tion of the C-terminal residue (Fig. 4B). All S
1
¢ and S
2
¢
residues interacting directly with RXPA381 are con-
served between ANCE and ACER except for the
aforementioned Val372 (ACER) and Thr364 (ANCE)
(Table 5). The molecular dynamic simulations suggest

that the methyl groups of the two alanines of
RXPA381 pack closely with Val372 of ACER, whereas
in ANCE, the methyl group of the terminal alanine
residue is orientated away from Thr364, reinforcing
the importance of the hydrophobicity of the valine side
chain.
Discussion
We have characterized the effect of Cl
–
on ANCE
activity by determining the kinetic constants for the
hydrolysis of AI in the absence and presence of NaCl
(100 mm). The increased k
cat
⁄ K
m
observed at pH 7,
was entirely the result of a 3.5-fold lowering of the K
m
for AI. A similar level of enhancement was also
achieved in the absence of NaCl by changing the pH
conditions from 7 to 8, although in this case changes
in both the K
m
and k
cat
contributed to the increased
catalytic efficiency. Although these effects are signifi-
cant, they are modest compared with the activation by
NaCl of the AI-converting activities of the C-domain

of human ACE [3,32]. ACER hydrolyses AI extremely
slowly, an activity that is not stimulated by Cl
–
. Never-
theless, a strong effect of NaCl on the peptidase
activity of ACER was observed when either [Leu5]
enkephalinamide or [Met5]enkephalinamide was the
substrate.
Our observation that NaCl alters the affinity of
ANCE for AI suggests that the binding of Cl
–
induces
a conformational change in ANCE that influences the
hydrolysis of AI. The molecular structures of two Cl
–
-
binding sites (Cl1 and Cl2) are known from the struc-
ture of human tACE [27], but no Cl
–
anions were
identified in the crystal structure of ANCE [28]. The
Cl2 Cl
–
-binding site of tACE, 10 A
˚
from the catalytic
zinc, is closer to the active site than Cl1 and comprises
the side chains of Arg522, Trp220 and Tyr224. Com-
paring the structures of ANCE, ACER and tACE at
the Cl2 binding site suggests that ANCE and ACER

would not bind Cl
–
at the Cl2 site. The substitution of
Pro519 in tACE by a glutamate in both ANCE and
ACER results in the carboxylic acid of this residue
residing in the space occupied by Cl
–
in the tACE crys-
tal structure [26].
The Cl1 binding site of tACE lies 20 A
˚
from the
catalytic zinc and involves three contacts, Arg186,
Trp485 and Arg489. Whereas Arg489 is conserved,
Arg186 and Trp485 of tACE are replaced by Tyr170
and Phe469 in ANCE. It has been proposed that the
Arg fi Tyr substitution may result in a Cl
–
-binding
site more similar to the ACE Cl2 binding site [26].
Although the Trp fi Phe substitution is expected to
reduce the affinity for Cl
–
, it is possible that the Cl1
site in ANCE may still bind the anion and that this
interaction is responsible for our observed increase in
affinity of ANCE for AI. In ANCE, the potential Cl1
binding site is adjacent to the peptide backbone of
Lys495, which our modelling, together with recent site-
directed mutagenesis studies on human ACE [33],

suggest direct interactions between Lys495 and the
C-terminus of the peptide substrate (Fig. 3). The pres-
ence of a Cl
–
ion at this site may have a stabilizing
effect on binding certain substrates.
In the N-domain of human ACE, and in ACER,
the Cl1 site is altered by the replacement of Arg186 of
tACE with His164 and His177, respectively, making it
unlikely that Cl
–
will bind at this position in both
these enzymes [26]. However, there is a possibility that
an alternative Cl
–
-binding site exists in the N-domain
of human sACE, as the R500Q mutant of the human
ACE N-domain, which removes the Cl2 site, responds
to 20 mm NaCl by a twofold increase in affinity for
AI [32]. The strong NaCl-induced activation of ACER
activity towards the amidated enkephalin substrates
and the unlikely involvement of the Cl1 and Cl2 sites
in this effect suggest that a different anion site may
also be present in ACER. A similar proposal for a
Cl
–
-binding site, distinct from the two identified in
tACE, has been put forward to explain the
Cl
–

-enhanced carboxypeptidase activity of human
ACE2 [29]. The lack of understanding of the molecu-
lar mechanism by which Cl
–
influences the catalytic
activity of ACEs is illustrated by the recent characteri-
zation of ACE from the leech Theromyzon tessulatum
[34]. The residues forming both Cl1 and Cl2 in tACE
are absolutely conserved in the leech enzyme, suggest-
ing that this ACE would, like human C-domain, be
strongly activated by NaCl. However, the enzyme
when expressed in mammalian cells responds with only
modest activation (twofold) of the hydrolysis of Hip-
His-Leu by NaCl with an optimal Cl
–
concentration
of 50 mm, and, thus, resembles the N-domain rather
than the C-domain of human ACE.
All the BK peptides used in this study were cleaved
by both ANCE and ACER, although ANCE was
invariably the more efficient enzyme, displaying
k
cat
⁄ K
m
values 30–100-fold greater than those obtained
with ACER. Our model of ANCE with either BK or
[Thr6]BK docked in the substrate channel suggests
Structure-activity of Drosophila ACEs R. J. Bingham et al.
368 FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS

that the acidic side chains of Asp360, as well as
Glu150, form favourable ionic interactions with the
positively charged C-terminal Arg of the peptides.
These residues are conserved in the human N-domain
and C-domain active sites (Table 5), both of which
efficiently cleave BK. However, Asp360 of ANCE is
replaced with His368 in ACER, and this change in the
electrostatic charge in the S
2
¢ pocket is predicted to
reduce ionic interactions between ACER and the
guanidino group of the C-terminal arginine of the BK
peptides. This may explain why the K
m
values for the
hydrolysis of BK, Ile-Ser-BK, [Thr6]BK and [Tyr8]BK
by ACER are 20–75-fold higher than the correspond-
ing values for ANCE. The model also suggests that an
N-terminal b-turn centred on the residues Pro2-Pro3
of BK and [Thr6]BK allows the peptides to fit tightly
into the larger (N chambers) of the two active-site cav-
ities, which may explain why BK peptides bind with
much higher affinity to ANCE and ACER than AI.
BK adopts a similar conformation in models of BK
bound to human C-domain ACE (R. J. Bingham,
unpublished work), which would provide an explan-
ation for why BK is the physiological substrate that
displays the highest-affinity of any substrate of the
human enzyme [2].
The affinity of BK for ANCE is increased almost

fourfold by introducing an extra methyl group in
[Thr6]BK. It has been shown previously that [Thr6]BK
has a markedly different solution structure to BK [35]
and has a greater tendency to adopt an N-terminal
b-turn, which was also a consistent feature of our
molecular modelling. The dynamic structure difference
between BK and [Thr6]BK provides a possible explan-
ation for the difference in binding affinity of these two
BK peptides to ANCE.
MKRSRGPSPRR is structurally related to mamma-
lian BKs and was shown to be an excellent ANCE
substrate. In contrast, this peptide was resistant to
hydrolysis by ACER and did not compete with sub-
strate for the enzyme active site. The surface of the
ACER active site is predicted to be positively charged,
which would present an unfavourable electrostatic
environment for Arg ⁄ Lys-rich peptides attempting to
access the substrate-binding channel. In contrast, the
negative charges lining the ANCE substrate chan-
nel would be expected to favour interactions with
positively charged peptide substrates, especially
MKRSRGPSPRR, which has positive charges along
the length of the peptide.
RXPA380 (Cbz-Phew[PO
2
-CH]Pro-Trp-OH) is a
highly selective inhibitor of the C-domain of somatic
ACE, with the pseudo-proline and the tryptophan resi-
dues in the P
1

¢ and P
2
¢ positions of the inhibitor being
important for determining this selectivity [31]. For both
ANCE and ACER, it is clear that proline in the P
1
¢
position does not allow strong inhibitor–enzyme inter-
action, as the substitution of the P
1
¢ proline of
RXPA380 with alanine in RXPA384 (Cbz-Phew[PO
2
-
CH]Ala-Trp-OH) makes a much more potent inhibitor
of both ANCE and ACER. The proline in RXPA380
probably restricts the orientation of the P
2
¢ side chain to
an orientation that is less favourable for interactions in
the S
2
¢ pocket of ANCE. Of the 12 residues of the S
2
¢
subsite of C-domain ACE that are predicted to interact
with the RXPA380 in a model of the inhibitor–enzyme
complex [31], only eight are strictly conserved in the N-
domain, nine in ANCE and eight in ACER (Table 4).
The adjacent valines (Val955 and Val956) that help

form the S
2
¢ pocket of C-domain ACE appear to be
involved in binding the tryptophan side chain of
RXPA380. It has been proposed that replacement of
these two residues in N-domain ACE with polar serine
and threonine will limit favourable hydrophobic inter-
actions between inhibitor and enzyme [31]. RXPA380
inhibits ACER, albeit weakly, but not ANCE. Our
model of the ACER–RXPA380 complex shows the
inhibitor bound in a very similar orientation to that des-
cribed for C-domain ACE, with the side chain of
Val372 (equivalent to Val956 of C-domain ACE)
involved in ligand interaction at the S
2
¢ pocket. The
replacement of Val372 of ACER with the polar Thr364
in ANCE probably contributes towards the lack of
inhibitory activity of RXPA380. This supports the
hypothesis that the hydrophobicity of Val956 in C-
domain ACE and Val372 in ACER is important for
RXPA380 selectivity. In our model, the larger side chain
of Gln266 restricts the space available for the large in-
dole ring of RXPA380 and would therefore contribute
together with Thr364 towards hindrance of RXPA380
binding to ANCE. In contrast, Thr858 of C-domain
ACE is replaced by the smaller Ser275, and ACE
Val956 is conserved as Val372 in ACER, which is con-
sistent with the inhibition of ACER by RXPA380.
RXPA381, which has alanine in both the P

1
¢ and P
2
¢
positions, inhibits both ANCE and ACER, but displays
100-fold selectivity in favour of ACER. This selecti-
vity is consistent with the observation that RXP407
(Ac-Asp-Phew[PO
2
-CH]Ala-Ala-NH
2
) and Ac-Asp-
Phew[PO
2
-CH]Ala-Ala-OH with a P
1
¢ and a P
2
¢ alanine
are also selective inhibitors of ACER [17]. The side
chain of Gln266 of ANCE, which forms the back of the
S
2
¢ site, is too distal (8 A
˚
) to interact with the P
2
¢ side
chains of RXPA381 and RXP407, and therefore will
not influence the binding of these less bulky inhibitors.

The unexpected result that ACER is inhibited by
both an N-domain-selective and a C-domain-selective
R. J. Bingham et al. Structure-activity of Drosophila ACEs
FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS 369
inhibitor demonstrates the dangers of classifying ACEs
as either N-domain-like or C-domain-like. Molecular
models of inhibitors complexed with ANCE and ACER
have suggested structural explanations for these obser-
vations and provided new insights into how structural
diversity in the ACE substrate channel can lead to
important differences in enzymatic properties. In addi-
tion, our models of BK docked at the ACE active site
have provided an explanation for the evolutionarily
conserved tight binding of this substrate to ACE.
Experimental procedures
Enzyme substrates and inhibitors
Peptides were purchased from Sigma-Aldrich (Poole,
Dorset, UK). RXPA380 (Cbz-Phew[PO
2
-CH]Pro-Trp-OH),
RXPA381 (Cbz-Phew[PO
2
-CH]Ala-Ala-OH), RXPA384
(Cbz-Phew[PO
2
-CH]Ala-Ala-OH) were synthesized as des-
cribed previously [8,31]. Abz-YRK(Dnp)P was a gift
from Professor Adriana K. Carmona, Department of Bio-
physics, Division of Nephrology, Escola Paulista de Medici-
na, Universidade Federal de Sao Paulo, Sao Paulo, Brazil.

Expression and purification of recombinant ANCE
and ACER
Recombinant ANCE and ACER were produced by expres-
sion in Pichia pastoris, as described previously [20,25]. Secre-
ted ANCE and ACER were purified to homogeneity from
the culture medium by using a combination of hydrophobic
interaction and ion-exchange chromatography. (NH
4
)
2
SO
4
was added to the culture media to a final concentration of
1.5 m, and, after centrifugation and filtration (0.2 lm pore
size; Minisart, Sartorius Ltd, Epsom, Surrey, UK), the cul-
ture media were applied to a column (12 cm · 2.6 cm)
packed with Phenyl-Sepharose Fast Flow 6 (Amersham
Biosciences, Chalfont St Giles, Buckinghamshire, UK)
pre-equilibrated with 1.5 m (NH
4
)
2
SO
4
⁄ 20 mm Tris ⁄ HCl,
pH 8.0. Protein was eluted with a decreasing gradient of
(NH
4
)
2

SO
4
(1.5–0 m; over 500 mL; flow rate of 5 mLÆmin
)1
)
and monitored using a UV detector set at 280 nm. Protein-
containing fractions were pooled and dialysed against
20 mm Tris ⁄ HCl, pH 8.0, before being applied to an ion-
exchange column (HiTrap Q HP, 5 mL bed volume; Amer-
sham Biosciences). Protein was eluted using a 200 mL gradi-
ent of increasing concentration of NaCl (0–1 m), at a flow
rate of 5 mLÆmin
)1
. Fractions containing enzyme activity,
determined using Hip-His-Leu as the substrate [36],
were pooled and dialysed against 100 mm Tris ⁄ HCl
(pH 7.0) ⁄ 50 mm NaCl ⁄ 10 lm ZnCl
2
, before being concen-
trated to 1 mg protein per ml of buffer using a centrifugal
concentrator (Microsep 10k; Pall Life Sciences, Portsmouth,
Hampshire, UK). The final protein concentration was
determined by absorbance at 280 nm. Cl
–
-free protein was
produced by dialysing 1 mL protein solution (1 mgÆmL
)1
)
against 5 L MilliQ water for 24 h followed by dialysis
against 100 mm Hepes (pH 8.0) ⁄ 10 lm ZnSO

4
for 24 h.
Enzyme assays
Dipeptidyl carboxypeptidase activity towards peptide sub-
strates was determined by HPLC quantification (214 nm) of
the reaction products (AII for the hydrolysis of AI; Phe-Arg
for the hydrolysis of BK, Ile-Ser-BK and [Thr6]BK; Tyr-
Arg for the hydrolysis of [Tyr8]BK; MKRSRGPSP for the
hydrolysis of MKRSRGPSPRR; Tyr-Gly-Gly, Phe-Leu-
amide and Met-Leu-amide for [Leu5]enkephalinamide and
[Met5]enkephalinamide; Tyr-Gly-Gly, Phe-Leu and Met-
Leu for [Leu5]enkephalin and [Met5]enkephalin). Unless
otherwise stated, the reactions were carried out at 35 °Cin
100 mm Hepes (pH 8.0) ⁄ 50 mm NaCl ⁄ 10 lm ZnSO
4
in a
final volume of 20 lL for AI and larger volumes (200 lL
to 1 mL) for BK and BK-related peptides. Reactions were
stopped by either addition of trifluoroacetic acid to a final
concentration of 2.5% or, for larger volumes, immersion in
boiling water for 5 min. HPLC analysis required different
reverse-phase columns and elution conditions to achieve
peptide separation. The products of AI, MKRSRGPSPRR,
and BK hydrolysis were resolved using a Phenomenex
Jupiter C18 (5 lm particles, 250 · 4.6 mm; Phenomenex,
Macclesfield, Cheshire, UK) column, whereas the separation
of BK 1–5 and BK 1–7 required a SuperPac Pep-S column
(5 lm particles, 250 mm · 4 mm; Amersham Biosciences).
The following elution gradients of acetonitrile in 0.1% tri-
fluoroacetic acid at a flow rate of 1 mLÆmin

)1
were used:
15–36% acetonitrile over 14 min for AII; 6–24% acetonitrile
over 22 min for Phe-Arg and MKRSRGPSP; 6–18% aceto-
nitrile for BK 1–5 and BK 1–7 over 20 min; 0–24% acetonit-
rile over 20 min for the separation of Tyr-Gly-Gly, Phe-Leu,
Met-Leu, Phe-Leu-amide and Met-Leu-amide. Identification
of peptides by MS was performed using a Q-Tof MS ⁄ MS
instrument. Hip-His-Leu hydrolysis was assayed as des-
cribed previously [36].
The kinetics of inhibition of ANCE and ACER by BK,
BK-related peptides and phosphinic acid inhibitors were
determined by measuring the effects on initial rates of
hydrolysis of Abz-YRK(Dnp)P (5 lm) in 100 mm Hepes,
pH 8.0, 50 mm NaCl and 10 lm ZnSO
4
(final reaction vol-
ume, 100 lL). ANCE and ACER hydrolysed Abz-
YRK(Dnp)P, a fluorogenic substrate based on the structure
of N-acetylSDKP [7], with K
m
values of 6.64 ± 1.1 lm and
4.60 ± 1.4 lm, respectively. The reactions were performed
at 20 °C in 96-well black plastic plates (Corning Life
Sciences, High Wycombe, Buckinghamshire, UK) using a
Victor
2
fluorimeter (PerkinElmer
TM
, Turku, Finland) to

quantify the rate of increase in fluorescence (k
em
430 nm
and k
ex
340 nm). The reaction was started by adding the
Structure-activity of Drosophila ACEs R. J. Bingham et al.
370 FEBS Journal 273 (2006) 362–373 ª 2005 The Authors Journal compilation ª 2005 FEBS
substrate to the enzyme in 100 mm Tris ⁄ HCl
(pH 7.0) ⁄ 100 mm NaCl ⁄ 10 lm ZnCl
2.
Kinetic parameters and IC
50
values were calculated using
nonlinear regression curve-fitting programs (figp; Biosoft,
Cambridge, UK). Error values are standard deviations of
the parameters calculated from the fitted curve by figp. The
K
i
of inhibition of ANCE by [Thr6]BK was determined by
measuring the kinetics of Abz-YRK(Dnp)P hydrolysis in
the presence of 0, 10, 20, 50 and 80 nm [Thr6]BK.
Molecular modelling
The model of D. melanogaster ACER was generated in
swiss-model [37] using the first approach mode and the crys-
tal structure of ANCE as a template (Protein DataBank
accession code 1J36). The zinc atom was manually posi-
tioned, co-ordinated by His375, Glu376 and His379, which
were deduced to be the co-ordinating residues by sequence
alignment. The co-ordinates of BK and [Thr6]BK were gen-

erated in pymol (), and manually posi-
tioned into the binding channel of ANCE and ACER using
the molecular visualization program O. The peptide was
aligned such that the carboxy group of the scissile peptide
bond was orientated towards the zinc according to the pro-
posed catalytic mechanism [27]. The large N-chamber and
C-chamber readily allowed positioning of the peptide with
minimal steric clashes. The model was then solvated with
explicit water molecules in a 20 A
˚
sphere centred on the pep-
tide. This model was improved by energy minimization and
molecular-dynamics simulations using the ds Modelling soft-
ware (Accelrys, San Diego, CA, USA). All energy calcula-
tions were performed using the CHARM22 force field, and
were restricted to the 20 A
˚
sphere centred on the peptide.
The nonbonded cut-off was set to 12 A
˚
. Initial optimization
was performed by two stages of energy minimization, firstly
500 steps of a conjugate gradient minimization, followed by
1000 steps using the adopted basis Newton–Raphson algo-
rithm. This was followed by heating to and equilibrium at
300 K before a 1000-step molecular-dynamics simulation
with time steps of 0.001 ps. Co-ordinates of RXPA380 were
kindly provided by Philippe Cuniasse, Departement d’Etudes
et d’Ingenierie des Proteines, Commissariat a l’Energie
Atomique, CE-Saclay, Gif-Sur-Yvette, France. Co-ordinates

of RXPA381 were generated in pymol. These co-ordinates
were then superimposed on to ANCE and ACER assuming a
similar binding orientation to ACE C-domain. This model
was then solvated with explicit water molecules in a 20 A
˚
sphere centred on the peptide and then subjected to the
molecular modelling scheme described above.
Acknowledgements
We thank Adriana K. Carmona (Universidade Federal
de Sao Paulo) for ACE substrates and Pam Gaunt
(University of Leeds) for technical expertise, Alison
Ashcroft (University of Leeds) for mass spectrometry,
Philippe Cuniasse (Commissariat a l’Energie Atomi-
que, CE-Saclay) for the pdb file of RXPA380, and
Pierre Corvol, Tracy Williams and Xavier Houard
(College de France, Paris) for Pichia expressing ANCE
and ACER. We acknowledge the support of the Bio-
technology and Biological Sciences Research Council
through a studentship to R.J.B. and a grant to A.D.S.
and R.E.I. (No. 89 ⁄ S19378).
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