©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Hemoregulatory Peptide N-Acetyl-Ser-Asp-Lys-Pro Is a Natural and Specific Substrate of the N-terminal Active Site of Human Angiotensin-converting Enzyme (*)

(Received for publication, August 7, 1994; and in revised form, November 7, 1994)

Anne Rousseau (§) Annie Michaud (1) Marie-Thérèse Chauvet (1) Maryse Lenfant (§) Pierre Corvol (1)(¶)

From the Centre National de la Recherche Scientifique, Institut de Chimie des Substances Naturelles, 91198 Gif-sur-Yvette, France and the Institut National de la Santé et de la Recherche Médicale, Unit 36, College de France, 3 rue d'Ulm 75005 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 µMbullets, 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.


INTRODUCTION

Angiotensin I-converting enzyme (ACE) (^1)(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. (^2)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.


EXPERIMENTAL PROCEDURES

Enzymes

Three purified human recombinant ACE proteins have been studied from stably expressed CHO cells: wild-type membrane-bound recombinant ACE, which contains the two intact functional domains, and two full-length ACE mutants containing only one intact functional domain by the substitution of the two zinc-binding histidyl residues of either the C- or the N-domain by lysyl residues (ACE, ACE). The construction of the ACE cDNA containing the entire coding sequence for human endothelial ACE and of the two ACE mutants, and their expression in CHO cells has already been described(13) . The purification of the corresponding proteins was performed according to Beldent et al.(27) , where the chromatographic steps have been replaced by centrifugation on an Ultrafree-MC membrane 100,000 PMNL (Millipore Corp.).

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.

Peptide

The tetrapeptide AcSDKP (purity >95%) was generously supplied by IPSEN-Biotech (Paris, France). The radiolabeled N-acetyl-Ser-Asp-[4-^3H]Lys-Pro ([^3H]AcSDKP), specifically tritiated on the lysyl residue, was prepared as described previously (purity >99% determined by radiochromatography HPLC)(31) . The specific radioactivity was about 90 Ci/mmol (3247 GBq/mmol).

Standard Enzymatic Assays

The enzymatic assays were performed in triplicate, at 37 °C, in 100 mM Tris-maleate buffer, pH 7.0, 10 µM ZnSO(4), 50 mM NaCl (buffer A). The kinetics of AcSDKP hydrolysis were determined by incubating enzymes (0.4 nM wild-type ACE, 0.3 nM ACE, and 9 nM ACE) with AcSDKP over a concentration range of 0.35-40 µM, added to [^3H]AcSDKP (5 µCi). The reaction was stopped by freezing on dry ice, and samples were then analyzed. AcSDKP (4 mM), Lys-Pro (KP) (2 mM) and lysine (1 mM) were added as carriers to the incubation media. To evaluate the radioactivity specifically associated with each peptide or amino acid component, the mixture was submitted to high voltage paper electrophoresis analysis, as described elsewhere(32) , a technique that appeared to be, in this case, as sensitive and accurate as HPLC analysis. Detection was performed with 0.1% ninhydrin in acetone. [^3H]AcSDKP and [^3H]KP were characterized by their respective migration distance, and the radioactivity associated with each spot was measured using an automatic beta scintillation counter. The average recovery of this technique was around 90%. For each substrate concentration, the percentage of AcSDKP hydrolysis (<10%) was then calculated, and the initial velocity of AcSDKP hydrolysis (V(0)) for the three recombinant enzymes was determined. Potential inhibition of ACE enzymatic activity by the product KP was investigated using Hip-His-Leu as substrate in the assay described above.

pH Dependence and Chloride Activation of AcSDKP Hydrolysis

The pH dependence of AcSDKP hydrolysis by wild-type ACE and ACE mutants was studied by incubating AcSDKP (100 nM) with wild-type ACE (2.5 nM), ACE (0.9 nM), and ACE (10 nM). Incubations were carried out at 37 °C, in 100 mM Tris-maleate buffer, 10 µM ZnSO(4), 50 mM NaCl, over a pH range of 5.0-9.0. Assays were also performed in 100 mM Hepes (pH 6.8-8.0) or potassium phosphate buffer (pH 5.5-8.0), 10 µM ZnSO(4), 50 mM NaCl. The reaction was stopped after a 10-min incubation time as described above.

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(4).

Inhibition of AcSDKP Hydrolysis by ACE Monoclonal Antibody

Monoclonal antibodies raised against wild-type human ACE are able to discriminate between the two homologous domains of ACE (33) . mAb i(2)H(5) (a gift from Dr S. Danilov, Institut National de la Santé et de la Recherche Médicale, Unit 367, Paris) raised against purified human lung ACE (34) recognizes epitopes in the N-terminal domain of ACE and inhibits the enzymatic activity of the N-terminal active site only(33) . The inhibitory potency of mAb i(2)H(5) toward wild-type ACE (2 nM), ACE (2 nM), and ACE (19 nM) was determined by establishing dose-dependent inhibition curves. The three forms of recombinant ACE and varying concentrations of mAb i(2)H(5) (1:1, 5:1, 50:1, and 250:1 molar ratios) were incubated overnight at 4 °C before the addition of AcSDKP (5 µM for wild-type ACE and ACE and 0.5 µM for ACE) to determine residual enzyme activity. The reaction was stopped after a 1-h incubation at 37 °C. Incubations in the absence of antibody were performed under the same conditions. Control incubations were carried out in the presence of: 1) antibody at the highest concentration, to study possible interactions between the peptide and the antibody, and 2) ACE with a monoclonal antibody (directed against angiotensinogen) at high concentration, to test nonspecific inhibition of ACE activity.

Inhibition of AcSDKP and Hip-His-Leu Hydrolysis by Various ACE Inhibitors

The potency of captopril (gift from Bristol Myers Squibb) and lisinopril (gift from Merck Sharp and Dohme) for the inhibition of AcSDKP hydrolysis by recombinant wild-type and mutant forms of ACE was determined by establishing dose-dependent inhibition curves at equilibrium. As tested compounds were competitive slow binding inhibitors of ACE, wild-type ACE (2 nM), ACE (2 nM), and ACE (20 nM) were preincubated with the different inhibitors for 30 min at 37 °C in buffer A, over a range of inhibitor concentrations around the IC (concentration of inhibitor needed to produce 50% inhibition). A preincubation of ACE without inhibitor was performed in duplicate under the same conditions. Reactions were initiated by the addition of 5 µM AcSDKP (corresponding to 0.15 times K(m)) for wild-type ACE and ACE and 0.5 µM AcSDKP for ACE, and reactions were terminated after 1 h. The product [^3H]KP was quantified as described above, and IC values were determined.

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(4). Residual free enzyme activity was determined by the addition of 500 µM Hip-His-Leu, corresponding to 0.25 times K(m). 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(i)(15) .


RESULTS

[^3H]AcSDKP was incubated with the recombinant wild-type and mutant forms of ACE, and the radioactivity associated with unhydrolyzed [^3H]AcSDKP and with the potential radioactive products [^3H]KP and [^3H]K was determined. The radioactivity profile indicated that in all cases a unique radiolabeled metabolite was generated, which corresponded to the dipeptide [^3H]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).

pH Dependence and Chloride Activation of AcSDKP Hydrolysis

The pH dependence of AcSDKP hydrolysis by the three recombinant forms of ACE was studied over a pH range of 5.0-9.0. Although the three enzymes exhibited an optimal activity at the same pH value of 6.5, they displayed different activity profiles over this pH range (Fig. 1): the pH dependence of ACE activity was shifted toward a lower pH compared with ACE (at pH 8.0, ACE and ACE activities were 50 and 25% of their optimal activities, respectively). In addition, the pH dependence curve of the wild-type ACE activity exhibited a diphasic profile following, at pH >7.0, the profile curve of ACE and, at lower pH, those of both ACE and ACE (Fig. 1, inset). Similar results were obtained in Hepes buffer with the three recombinant forms of ACE, whereas a complete inhibition of the three recombinant enzymes was observed in phosphate buffer (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 (circle) 10 nM ACE (times), and 2.5 nM wild type (bullet) 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(4), 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`(a)) 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(4), pH 7.0, buffer. AcSDKP was incubated independently with 0.6 nM ACE (circle), 26 nM ACE (times) and 2 nM wild-type ACE (bullet). The velocity (V(o)) of each reaction was determined and the optimal velocity (V) was calculated in each case. The ratio V(o)/V was plotted against NaCl concentration.



Kinetic Parameters of AcSDKP Hydrolysis

The K(m) values at 50 mM NaCl calculated from Lineweaver-Burk plots (Fig. 3, A and B), by wild-type ACE, ACE, and ACE were 41, 31, and 39 µM, respectively (Table 1). The results are the means of three independent determinations, which varied by 10-30%. A slight difference of the K(m) values was observed, but the k values differ markedly (Table 1). The wild-type ACE and ACE exhibit similar k values, whereas ACE hydrolyzes AcSDKP at a much slower rate. The k/K(m) value obtained for ACE is similar to that of the wild-type enzyme. The same study was carried out in the presence of 300 mM NaCl for ACE. The K(m) and k/K(m) values determined remained similar to those determined at 50 mM NaCl and were 10 µM and 0.01 µMbullets, respectively.


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(4), 50 mM NaCl, pH 7.0, buffer in a final volume of 90 µl. Initial rates (V(o)) of AcSDKP hydrolysis were calculated from [^3H]Lys-Pro dipeptide formed (<10%) for each substrate concentration (S), and the ratio (E)/V(o) was plotted against 1/S for (A) ACE (circle) and wild-type ACE (bullet) and (B) ACE (times). The inset shows a replot on a larger scale of the intersections of the axes.





Inhibition by an ACE Monoclonal Antibody

As shown in Table 2, the ACE mAb i(2)H(5) was significantly more effective in inhibiting AcSDKP hydrolysis by ACE than by ACE. A 50% inhibition was observed at a mAb i(2)H(5)/ACE molar ratio of 5:1, and only a 12% inhibition was obtained at a mAb i(2)H(5)/ACE molar ratio of 250:1. The inhibitory potency of the mAb i(2)H(5) on wild-type ACE activity was close to that observed with ACE.



Inhibition of AcSDKP Hydrolysis by ACE Inhibitors and Comparison with Inhibition of Hip-His-Leu Hydrolysis

The inhibitory potencies of ACE inhibitors toward AcSDKP hydrolysis by the recombinant enzymes were determined. Captopril and lisinopril appeared to be potent inhibitors of AcSDKP hydrolysis by wild-type ACE and ACE; IC values were about 2 nM, whereas for ACE IC values were about 30 nM.

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(i) 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.


DISCUSSION

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(4) 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(4) ions and AcSDKP. (^3)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`(a) = 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`(a) = 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(m) 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(m) 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(2)H(5) 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(2)H(5), 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(m) 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(m) 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.


FOOTNOTES

*
This work was supported by the Ligue Nationale contre le Cancer, by the Institut National de la Santé et de la Recherche Médicale, and by a grant from the Bristol-Myers-Squibb Institute for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: College de France, 3 rue d'Ulm, 75005 Paris France. Tel.: 33-1-44-27-16-75; Fax: 33-1-44-27-16-91.

(^1)
The abbreviations used are: ACE, angiotensin-converting enzyme; LH-RH, luteinizing hormone-releasing hormone; CHO, Chinese hamster ovary; Hip, hippuryl; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; AcSDKP, N-acetyl-seryl-aspartyl-lysyl-proline.

(^2)
A. Rousseau, A., Michaud, M.-T. Chauvet, M. Lenfant, and P. Corvol, unpublished results.

(^3)
D. Guillaume, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. E. Deschamps de Paillette for constant encouragements. We are grateful to Florent Soubrier for providing human ACE cDNA clones and Lei Wei for CHO cells transfected with wild-type and mutant cDNA ACE. We especially thank Tracy Williams for critical reading of the manuscript and Nicole Braure for assistance in manuscript preparation.


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