(Received for publication, August 7, 1994; and in revised form, November 7, 1994)
From the
Angiotensin I-converting enzyme (ACE) is a zinc-dipeptidyl
carboxypeptidase, which contains two similar domains, each possessing a
functional active site. Respective involvement of each active site in
the degradation of the circulating peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a negative
regulator of hematopoietic stem cell proliferation, was studied by
using wild-type recombinant ACE and two full-length mutants containing
a single functional site. Both the N- and C-active sites of ACE exhibit
dipeptidyl activity toward AcSDKP, with K values of 31 and 39 µM, respectively. However,
the N-active site hydrolyzes the peptide 50 times faster compared with
the C-active site, with k
/K
values of 0.5 and 0.01
µM
s
,
respectively. The predominant role of the N-active site in AcSDKP
hydrolysis was confirmed by the inhibition of hydrolysis using a
monoclonal antibody specifically directed against the N-active site.
The N-domain specificity for AcSDKP will aid the identification of
specific inhibitors for this domain. This is the first report of a
highly specific substrate for the N-active site of ACE, with kinetic
constants in the range of physiological substrates, suggesting that ACE
might be involved via its N-terminal active site in the in vivo regulation of the local concentration of this hemoregulatory
peptide.
Angiotensin I-converting enzyme (ACE) ()(peptidyl
dipeptidase A, kininase II, EC 3.4.15.1) is a zinc-dipeptidyl
carboxypeptidase, which also displays endopeptidase activity on certain
substrates. The primary specificity of ACE is to cleave C-terminal
dipeptides from oligopeptide substrates with a free C terminus in the
absence of a penultimate proline residue or a terminal dicarboxylic
amino acid. ACE cleaves the C-terminal dipeptide from angiotensin I to
produce the potent vasopressor octapeptide, angiotensin II(1) ,
and inactivates bradykinin by the sequential removal of two C-terminal
dipeptides(2) . The endopeptidase activity of ACE is observed
with substrates that are amidated at their C termini where the enzyme
cleaves a C-terminal dipeptide amide and/or a C-terminal tripeptide
amide(3, 4) . An unexpected activity of ACE is
observed on luteinizing hormone-releasing hormone (LH-RH) where ACE can
cleave not only the C-terminal tripeptide amide but in addition the
N-terminal tripeptide (5) .
There are two ACE isoforms: a somatic form of about 150-180 kDa found in endothelial, epithelial, and neuroepithelial cells and a smaller isoform of 90-110 kDa present only in male germinal cells. The somatic form of ACE is derived from a duplicated ancestral gene (6, 7) and is composed of two highly homologous domains called N- and C-domains, each comprising an active site that faces the extracellular surface of the cell(8, 9) . Molecular cloning of the germinal form of ACE has revealed that it corresponds to the C-domain of somatic ACE with the exception of a short specific N-terminal sequence and therefore contains a single active site(10, 11, 12) .
It was
established previously that both putative active sites of the somatic
enzyme were functional by constructing a series of ACE mutants (13) . Further studies demonstrated that both domains exhibit
similar catalytic activities toward the substrates angiotensin I,
bradykinin, and substance P(14) . However, there are some
differences between the two functional domains of ACE. 1) The two
active sites are differently activated by chloride ions. The C-terminal
domain activity is highly dependent on chloride concentration, whereas
the N-terminal domain is still active in the absence of chloride and is
fully activated at relatively lower chloride
concentrations(13, 14) . 2) A number of specific ACE
inhibitors display different potencies toward the two active
sites(15) . 3) The N-active site is preferentially involved in
the N-terminal endopeptidase cleavage of LH-RH(14) . However,
the K for this cleavage is rather high,
and the catalytic efficiency is low; therefore, it is unlikely that
this cleavage occurs in vivo. In fact, no specific substrate
for the N-domain has been identified to date.
The tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), isolated from
fetal calf bone marrow(16) , is involved in the control of
hematopoietic stem cell proliferation by preventing their recruitment
into S-phase(17) . AcSDKP appears to exert this function by
blocking the action of a stem cell-specific proliferation stimulator (18) and acts selectively on quiescent progenitors. The
phase-specific anti-cancer drugs act on cycling cells and thus do not
select between malignant cells and normal progenitors undergoing
cytotoxic treatment. Therefore, administration of AcSDKP in conjunction
with cytotoxic therapy offers a potential therapeutic application in
selectively maintaining normal progenitors in the quiescent
state(19, 20) . Co-localization of AcSDKP and ACE in
humans has been observed in plasma (21, 22, 23) and in circulating mononuclear
cells(24, 25) . Moreover, in mice, the presence of
both AcSDKP and ACE was detected in bone marrow cells. ()These results suggest that ACE might be involved in the in vivo regulation of the local concentration of the
hemoregulatory peptide AcSDKP. The inhibitory effect of AcSDKP depends
on its half-life. Recently, it was shown that AcSDKP circulates in
plasma and that ACE is involved in vitro in the initial and
determinant catabolic step of AcSDKP hydrolysis(26) .
In the
present study, the respective activities of the wild-type
membrane-bound recombinant human ACE and of two full-length ACE mutants
in the degradation of AcSDKP were determined. The mutants contain one
of the two active sites inactivated by substitution of the two
zinc-binding histidines by lysyl residues and are referred to here as
ACE and ACE
, corresponding to the
C- and N-functional domains of ACE, respectively. This study reveals
that the activity of wild-type ACE on AcSDKP hydrolysis is entirely
attributable to its N-active site, a result confirmed by the complete
inhibition of this hydrolysis by a monoclonal antibody specifically
directed against the N-active site. Finally, the potency of specific
ACE inhibitors on AcSDKP hydrolysis was evaluated and compared with
that obtained on hydrolysis of a specific substrate of the C-active
site of the enzyme.
Altogether our data suggest that the N-terminal active site of ACE is specifically involved in the catabolism of AcSDKP, thus identifying the first potential physiological and specific substrate for this catalytic domain.
The enzymatic activities of the three recombinant enzymes were assayed using hippuryl-histidyl-leucine (Hip-His-Leu) as substrate as described by Cushman and Cheung (28) and quantified as described previously(29) . ACE concentrations were quantified by direct RIA (30) or were deduced from their enzymatic activities.
The effect of various chloride concentrations
(0-600 mM) on AcSDKP (100 nM) hydrolysis was
studied by incubation with wild-type ACE (2 nM),
ACE (0.6 nM), and ACE
(26 nM) in 100 mM Tris-maleate buffer, pH 7.0,
10 µM ZnSO
.
In order to compare the
effects of these inhibitors on two highly different ACE substrates with
regard to the activities of the N- and C-domains of ACE, the inhibition
of Hip-His-Leu hydrolysis was also studied. Wild-type ACE (0.01
nM), ACE (0.01 nM), and
ACE
(0.1 nM) were preincubated with various
concentrations of inhibitors for 30 min at 37 °C, in 100 mM potassium phosphate buffer, pH 8.3, 300 mM NaCl, and 10
µM ZnSO
. Residual free enzyme activity was
determined by the addition of 500 µM Hip-His-Leu,
corresponding to 0.25
K
. Initial
velocities were measured during the first 5% of substrate hydrolysis,
and under these conditions the IC
can be considered to
correspond to the K
(15) .
[H]AcSDKP was incubated with the
recombinant wild-type and mutant forms of ACE, and the radioactivity
associated with unhydrolyzed [
H]AcSDKP and with
the potential radioactive products [
H]KP and
[
H]K was determined. The radioactivity profile
indicated that in all cases a unique radiolabeled metabolite was
generated, which corresponded to the dipeptide
[
H]KP (results not shown). No ACE inhibition by
the product Lys-Pro up to concentrations 10 times higher than that
generated during maximal AcSDKP hydrolysis was detected using
Hip-His-Leu as substrate (results not shown).
Figure 1:
pH
dependence of AcSDKP hydrolysis by recombinant forms of ACE. AcSDKP
(100 nM) was incubated with 0.9 nM ACE (
)
10 nM ACE
(
), and 2.5 nM wild type (
) in a final
volume of 90 µl. The hydrolysis was carried out at 37 °C, from
pH 5.0 to 9.0, in 100 mM Tris-maleate, 10 µM ZnSO
, 50 mM NaCl buffer. The ratio
(percentage of AcSDKP hydrolysis)/(ACE molarity) was plotted against
pH. The inset represents the percentage of AcSDKP hydrolysis
plotted against pH.
The chloride sensitivity of
AcSDKP hydrolysis by the wild-type and the two mutant forms of ACE was
then analyzed at pH 7.0 (Fig. 2). The activities of the
wild-type enzyme and of ACE were optimal at chloride
concentrations of around 50 mM and were inhibited at
supra-optimal chloride concentrations. An optimal activity of
ACE
was observed at chloride concentrations of
around 300 mM, although 70% of optimal activity was observed
at 50 mM NaCl (Fig. 2). The apparent activation
constants (K`
) for both the wild-type enzyme and
ACE
were 5 mM, whereas that for
ACE
was 29 mM.
Figure 2:
Chloride dependence of AcSDKP hydrolysis
by recombinant forms of ACE. AcSDKP (100 nM) hydrolysis was
evaluated at 37 °C over the range of NaCl concentrations of
0-600 mM, in 100 mM Tris-maleate, 10 µM ZnSO, pH 7.0, buffer. AcSDKP was incubated
independently with 0.6 nM ACE
(
), 26
nM ACE
(
) and 2 nM wild-type ACE (
). The velocity (V
) of
each reaction was determined and the optimal velocity (V
) was calculated in each case. The ratio V
/V
was plotted against
NaCl concentration.
Figure 3:
Lineweaver-Burk plots of AcSDKP hydrolysis
by recombinant forms of ACE. Enzyme assays were carried out at 37
°C, in 100 mM Tris-maleate, 10 µM ZnSO, 50 mM NaCl, pH 7.0, buffer in a final
volume of 90 µl. Initial rates (V
) of AcSDKP
hydrolysis were calculated from [
H]Lys-Pro
dipeptide formed (<10%) for each substrate concentration (S), and the ratio (E)/V
was
plotted against 1/S for (A) ACE
(
) and wild-type ACE (
) and (B)
ACE
(
). The inset shows a replot on
a larger scale of the intersections of the axes.
Since AcSDKP behaved as a specific
substrate of the N-active site, it was interesting to compare the
inhibitory potency of these ACE inhibitors using Hip-His-Leu as
substrate, which is almost exclusively cleaved by the C-active site.
The inhibitory potency of captopril was evaluated as 4 nM, 0.4
nM, and 2 nM for wild-type ACE,
ACE, and ACE
, respectively. K
values obtained for lisinopril were 0.5
nM, 7 nM, and 0.3 nM for wild-type ACE,
ACE
, and ACE
, respectively.
Captopril was more efficacious toward the N-active site than the
C-active site, whereas the reverse was observed for lisinopril.
Several lines of evidence have been reported previously that
indicate the involvement of human plasma ACE in the in vitro degradation of the tetrapeptide AcSDKP(26) , a negative
regulator of the hematopoietic stem cell proliferation(16) .
The present study was undertaken to determine precisely the
characteristics of AcSDKP degradation by ACE. Data indicate that
recombinant wild-type ACE and the two full-length N-active
(ACE) and C-active (ACE
) mutants
of ACE are able to cleave AcSDKP by a dipeptidase activity. However,
the N- and C-active sites were differently involved in AcSDKP
hydrolysis since they displayed markedly different kinetic parameters
and different responses to pH, chloride ions, and ACE inhibitors.
The pH dependence profile of AcSDKP hydrolysis was similar for the
three different recombinant enzymes with an optimum pH of 6.5. A strong
inhibition of all enzyme activities was observed in the presence of
PO ions, as observed previously for ACE
acting on two other synthetic substrates, Hip-Gly-Gly (35) and
para-nitrobenzyloxycarbonyl glycyltryptophylglycine(36) . A
nuclear magnetic resonance spectroscopy (NMR) study ruled out an
association between PO
ions and AcSDKP. (
)The inhibitory effect of phosphate ions on ACE activity is
not well understood and has not been observed for the substrates
Hip-His-Leu or angiotensin I(28, 37) . It may be
possible that there is a direct interaction of the monobasic form of
phosphate with the peptidic chain of the enzyme resulting in the
prevention of the hydrolysis of AcSDKP.
The hydrolysis of AcSDKP by
the three recombinant forms of ACE occurred slowly in the absence of
chloride ions and was activated by chloride addition. In the case of
the wild-type recombinant ACE, optimal activity was observed at a 50
mM chloride concentration, and an inhibition was observed at a
concentration above 100 mM. In this respect, AcSDKP, which
possesses a lysyl residue at the penultimate position, is analogous to
the classical ACE class II substrates (38) and can be
considered as one of those substrates with a chloride activation
constant value of K` = 5 mM. Similar
characteristics were also found for ACE
. Conversely,
ACE
hydrolyzes AcSDKP optimally at chloride
concentrations above 300 mM, and is characterized by an
activation constant of K`
= 29 mM which classifies AcSDKP as an ACE class III substrate for the
C-active site of ACE. A difference in the chloride sensitivity of the
two active sites of ACE has already been reported for angiotensin I
hydrolysis by the same recombinant enzymes(14) .
The K values of AcSDKP hydrolysis by the wild type and
the two ACE mutants were similar and revealed a strong affinity of
AcSDKP for the two active sites of ACE. However, a marked difference in
the catalytic efficiency of the two domains was observed and was
attributable to the differences in the k
values:
the ratio of k
/K
varied
over 50-fold when the hydrolyses by ACE
and
ACE
were compared. In addition, the catalytic
efficiency of the wild-type ACE is similar to that of
ACE
, an observation that is consistent with ACE
hydrolyzing AcSDKP by its N-active site. This hypothesis is also
supported by the weak chloride activation profile of the wild-type
enzyme for AcSDKP hydrolysis since the N-domain is much less sensitive
to chloride ions than the C-domain. Moreover, the anti-catalytic
activity of mAb i
H
was examined using AcSDKP as
substrate. This antibody recognizes epitopes specific to the N-terminal
active site (33) and can be used to investigate the respective
roles of each active site toward AcSDKP hydrolysis. The results show
that, in the presence of mAb i
H
,
ACE
activity was inhibited, whereas AcSDKP
hydrolysis by ACE
was not prevented. In addition,
the inhibition profile of the wild-type ACE was quite similar to that
of ACE
, further supporting the proposal that AcSDKP
cleavage is performed mainly by the N-active site of wild-type ACE.
It is interesting to compare the kinetic constants for the
hydrolysis of AcSDKP by the three recombinant forms of ACE with those
for the hydrolysis of two other natural substrates, angiotensin I and
LH-RH (Table 3). Angiotensin I is cleaved equally at 50 mM NaCl by the N- and the C-domains, and their combined activities
are equal to that of the wild-type enzyme(13) . LH-RH is
cleaved at its C terminus by both domains, but its N-terminal
tripeptide is cleaved 10 times faster by the N-domain than by the
C-domain. Besides the fact that the difference in the catalytic
efficiencies between the two domains is not large, the high K and the overall low catalytic efficiency of
recombinant forms of ACE for LH-RH hydrolysis indicated that this
peptide is an unlikely physiological substrate for ACE. Therefore, it
appears that AcSDKP is the first potential physiological substrate
specifically hydrolyzed by the N-terminal active site, with a
k
/K
value similar to that of
angiotensin I(13) . All of these results indicate important
functional differences between the subsites of the two active catalytic
sites of ACE despite their high degree of amino acid sequence homology.
These studies have demonstrated a distinct specificity for the N-domain
in the hydrolysis of AcSDKP, which is the shortest natural N-terminal
blocked substrate described for ACE. ACE hydrolyzes the physiological
substrates angiotensin I and bradykinin by a dicarboxypeptidase
activity of the two active sites. At present, it is not possible to
determine whether the N-active site of ACE acts on AcSDKP as an
endopeptidase (as in the case of the N-terminal endopeptidase cleavage
of LH-RH) or, more likely, via its classical peptidyl dipeptide
hydrolase activity since this peptide has a free C terminus.
The
effect of different ACE inhibitors was studied on the hydrolysis of two
substrates, one specifically hydrolyzed by the N-active site (AcSDKP)
and the other by the C-active site (Hip-His-Leu). In the case of AcSDKP
hydrolysis, the inhibitory potency of these inhibitors toward wild-type
ACE and ACE was similar but markedly different from
that against ACE
. In the case of Hip-His-Leu
hydrolysis, the inhibitory potencies observed for the wild-type ACE and
ACE
were closer than those obtained with
ACE
. This suggests that the relative potency of
these inhibitors is dependent on the specificity of the substrate for
each active site. This observation has practical consequences for the
discovery of inhibitors specifically directed against one of the two
active sites of ACE. In the case of the N-active site of the enzyme, in vitro hydrolysis of AcSDKP could be a useful tool for
screening inhibitors specifically directed against this active site.
The design of inhibitors of the N-active site of ACE and their administration to humans during cancer therapy could enhance the in vivo stability of AcSDKP and thus increase the capacity of AcSDKP to reversibly block the action of the hematopoietic stem cell proliferation stimulators(18, 39) , found in regenerating tissues treated with cytotoxic drugs used in chemotherapy(40) . Specific inhibitors of the N-domain could potentially enable the increase in physiological concentrations of AcSDKP without altering the activity of the C-domain on angiotensin I and bradykinin metabolism, thereby not interfering with the function of ACE in blood pressure and water and salt homeostasis.