Introduction

Summary Introduction Procedures Results Discussion References

ACE (peptidyl dipeptidase A; EC 3.4.15.1) is a zinc metalloenzyme anchored to the plasma membrane, with the bulk of its mass, including its active sites, exposed at the extracellular surface of the cell. The enzyme displays a wide tissue distribution in that it is expressed at the surface of endothelial, epithelial, and neuroepithelial cells (1). Two distinct isoforms of ACE are expressed in mammalian tissues: the predominant isoform (referred to here as somatic ACE) is composed of two highly similar domains (called N and C domains) with a molecular mass of 170 kDa (2). The other isoform is expressed exclusively in mature spermatids (germinal ACE); it is composed of a single catalytic domain with a molecular mass of 100 kDa and is identical to the C domain of somatic ACE with the exception of a short amino-terminal germinal ACE-specific sequence (3).

The primary specificity of the enzyme is to remove carboxyl-terminal dipeptides from the carboxyl terminus of an oligopeptide substrate, although other, atypical cleavages have been described (4, 5). The classic physiological substrates of ACE are Ang I, which is converted to the vasopressor Ang II by the removal of a single carboxyl-terminal dipeptide (6), and the vasodilatory peptide bradykinin, which is inactivated by the sequential removal of two carboxyl-terminal dipeptides (7). Thus, ACE plays a central role in blood pressure homeostasis, and a large number of highly potent and specific ACE inhibitors have been developed that are used as orally active drugs in the treatment of hypertension and congestive heart failure. The current generation of ACE inhibitors exert their action by coordinating the catalytically essential active site zinc ion and were designed on the basis of the known active site of another zinc metalloenzyme, carboxypeptidase A. However, a new generation of ACE inhibitors has been developed that have been synthesized with the aim of inhibiting not only ACE but also other zinc metallopeptidases involved in cardiovascular regulation. Such mixed inhibitors have been designed successfully for ACE-neutral endopeptidase (8) and for ACE-thromboxane synthase (9).

In addition to the well-established roles of ACE in Ang I and bradykinin metabolism, ACE has recently been demonstrated to be involved both in vitro (10) and in vivo (11) in the hydrolysis of AcSDKP, a regulatory factor of hematopoiesis. A competitive ACE inhibitor, captopril, has been shown to inhibit angiogenesis by being able to block neovascularization in vivo (12). Therefore, the physiological functions of ACE are not limited to its cardiovascular role, and ACE may be involved in hematopoietic stem cell regulation by constantly degrading AcSDKP, a natural circulating inhibitor of cell entry into S phase. Interestingly, AcSDKP is hydrolyzed 50-fold faster by the N domain-active site compared with the C domain and constitutes the only natural and specific substrate identified to date of the N domain of somatic ACE. This newly discovered function of ACE reveals that ACE inhibitors, which inhibit both the N and C domain-active sites of somatic ACE, can act on peptides involved in functions unrelated to blood pressure and water and salt homeostasis.

A series of ACE mutants containing only one intact domain were used to demonstrate that the N and C domains exhibit similar catalytic activities toward Ang I, bradykinin, and substance P (13). However, the N and C domains exhibit some interesting functional differences: (a) the two active sites are activated differently by chloride ions; in particular, the C domain-active site is far more sensitive to chloride concentration for the hydrolysis of some substrates, (b) the ACE N domain catalyzes both in vitro and in vivo AcSDKP degradation, and (c) the two domains of ACE interact differently with competitive inhibitors, and the chloride concentration has a more marked effect on inhibitor interaction with the C domain than with the N domain (14). These functional differences indicate that despite the high level of primary sequence homology between the N and C domains, structural differences do exist between the two active sites of somatic ACE, raising the possibility of designing or screening for ACE domain-specific inhibitors. The development of active site-specific inhibitors is of considerable interest: a specific inhibitor of the N domain would increase plasma AcSDKP levels without altering Ang I and bradykinin metabolism by the C domain. Such an inhibitor would be of potential value during chemotherapy in the protection of hematopoietic stem cells without affecting cardiovascular function.

To identify an ACE inhibitor that displays some selectivity for the inhibition of AcSDKP hydrolysis compared with that of Ang I, we used a range of specific ACE inhibitors to analyze the inhibition of hydrolysis of these two substrates by somatic ACE and two full-length ACE mutants that contain a single functional domain. The ACE inhibitors used in this study were chosen on the basis of their interaction with the inhibitor binding subsites of ACE as well as the coordination of the zinc ion or ions in the ACE active site or sites: captopril and fosinoprilat coordinate the active site zinc ion via their sulfydryl and phosphinyl groups, respectively, whereas lisinopril binds the zinc ion via its carboxyl group (Fig. 1). In addition, with a view to the large-scale screening of preexisting compounds for a N domain-specific inhibitor, we tested the inhibition (by using the same ACE inhibitors described above) of wild-type somatic ACE and the N and C domains using three synthetic substrates: (a) Hip-His-Leu, which mimics the carboxydipeptide of Ang I; this peptide is preferentially cleaved by the ACE C domain and has been routinely used for ACE inhibitor screening, (b) Hip-Lys-Pro, which mimics the carboxydipeptide of AcSDKP and as such is a potential specific synthetic substrate of the N domain, and (c) Hip-Ala-Pro, which mimics the dipeptide of captopril and is an example of a substrate whose hydrolysis is not chloride dependent (15).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the binding of inhibitors and substrates to the active site of ACE.

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Materials

Enzymes. Wild-type somatic ACE and the two ACE mutants containing only one functional active site were obtained through stable expression from CHO cells transfected with the appropriate ACE cDNA. The two ACE mutants were full-length enzymes with either the N or C domain catalytic site inactivated by substitution of the two zinc-binding histidyl residues by lysyl residues (ACE_K361,365 and ACE_K959,963). These mutants are hereafter referred to as the N and C domains, indicating the sole catalytically active domain. The construction of the ACE cDNAs and their expression in CHO cells have been previously described (16). The purification of the corresponding membrane-bound proteins was performed according to Beldent et al. (17), except that the chromatographic steps were replaced by ultrafiltration on an Ultrafree-MC membrane, nominal molecular mass limit, 100,000 D (Millipore, Bedford, MA).

Peptides. Hip-His-Leu, His-Leu, AcSDKP, Lys-Pro, Ala-Pro, and Ang I were purchased from Bachem (Bubendorf, Switzerland). Ang II and hippuric acid (Hip) were from Sigma Chemical (Poole, Dorset, UK). Hip-Lys-Pro and Hip-Ala-Pro were synthesized by Neosystem (Strasbourg, France). The purity of these last two peptides was greater than 90%.

Inhibitors. Captopril and fosinoprilat were a gift from Bristol-Myers Squibb (Princeton, NJ) and lisinopril was donated by Merck Sharp and Dohme (Paris, France).

Methods

Kinetic and inhibition studies of wild-type ACE and ACE mutants were performed using Hip-His-Leu, Hip-Lys-Pro, Hip-Ala-Pro, Ang I, and AcSDKP as substrates in the presence of different ACE inhibitors. For each enzyme and substrate, the optimal Cl concentration was first determined as described below.

The rate of hydrolysis of all substrates used was quantified by HPLC on a Waters (Milford, MA) apparatus consisting of 600 controllers, 486 absorbance detectors and 717 or 712 autosamplers WISP directed by a millennium chromatography manager.

Hip-Lys-Pro and Hip-Ala-Pro hydrolysis. Reactions were performed using 0.1-0.5 × 10⁹ M enzyme in 100 mM potassium phosphate, pH 8.0, 10 µM ZnSO₄, 20 mM or 100 mM NaCl, and 1 mg/ml BSA. Reactions were initiated by the addition of Hip-Lys-Pro or Hip-Ala-Pro in a total volume of 250 µl and incubated for 10 or 15 min at 37° to produce 5% substrate hydrolysis. Reactions were terminated by the addition of 12% H₃PO₄ (50 µl). Hip and Hip-Lys-Pro were resolved and quantified by isocratic reverse-phase HPLC on a 10-µm Nucleosil C18 column in 24% (v/v) acetonitrile, and 1 mM potassium phosphate- pH 3.0, at a flow rate of 1.0 ml/min. Retention time for Hip and Hip-Lys-Pro was 6.7 and 4.94 min, respectively, with a detection limit of 0.1 nmol at 228 nm. Hip and Hip-Ala-Pro were resolved and quantified as for Hip and Hip-Lys-Pro but in 15% (v/v) acetonitrile and 1 mM potassium phosphate, pH 3.0. Retention time for Hip and Hip-Ala-Pro was 8.8 and 12.0 min, respectively, with a detection limit of 0.1 nmol at 228 nm.

Ang I hydrolysis. Reactions were performed using 0.1-0.2 × 10⁹ M enzyme in 50 mM HEPES, pH 7.5, 50 mM NaCl, 10 µM ZnSO₄, and 1 mg/ml BSA and stopped by the addition of 2% H₃PO₄ (final concentration). Ang II and Ang I were resolved and quantified by isocratic reverse-phase HPLC in 18% (v/v) acetonitrile and 86 mM triethylammonium phosphate, pH 3.0, at a flow rate of 1 ml/min on a 3-µm Hypersil C18 column at 45°. Retention time of Ang II and Ang I was 3.4 and 7.0 min, respectively, with a detection limit of 0.5 nmol at 200 nm.

AcSDKP hydrolysis. Reactions were performed using 0.2-2 × 10⁹ M enzyme in 100 mM HEPES, pH 7.0, 50 mM NaCl, 10 µM ZnSO₄, and 1 mg/ml BSA and terminated by the addition of 0.1% trifluoracetic acid (final concentration). The rate of hydrolysis of AcSDKP was determined by measuring the production of Lys-Pro, which was resolved and quantified under isocratic conditions [water/acetonitrile (98:2), 0.1% trifluoracetic acid]. Unhydrolyzed AcSDKP was eluted and quantified by increasing the concentration of acetonitrile to 10%. The products were resolved by reverse-phase HPLC on a 5-µm Puresil C18 column at a flow rate of 1 ml/min. Retention time of Lys-Pro and AcSDKP was 4.7 and 13.2 min, respectively, with a detection limit of 0.2 nmol at 200 nm.

Enzyme inhibition assays. The potency of each inhibitor toward each form of ACE was determined by establishing dose-dependent inhibition curves at equilibrium. Because all the compounds tested are competitive slow-tight binding inhibitors, the preincubation time required to produce steady state equilibrium between enzyme and inhibitor was first determined. Preincubations were performed using 0.1 × 10⁹ M enzyme and 0.25-1 × 10⁹ M inhibitor at 37° for 0-120 min ,and reactions were initiated by the addition of substrate. A preincubation of 1 hr was required for fosinoprilat, whereas the inhibition by captopril and lisinopril was unchanged for a 0.5-2-hr preincubation. Enzyme activities in the absence of inhibitor did not change during control incubations of 3 hr, demonstrating enzyme stability for 3 hr at 37°.

9

9

9

9

9

9

9

11

8

9

5

	Results

Summary Introduction Procedures Results Discussion References

Kinetic studies of wild-type ACE and the ACE mutants were performed using Hip-His-Leu, Hip-Lys-Pro, Hip-Ala-Pro, Ang I and AcSDKP, as substrates. We tested the potential inhibitory action of the dipeptide products generated from the hydrolysis of the five substrates: Lys-Pro, Ala-Pro, and His-Leu. Inhibition of ACE activity was detected only at high concentrations of either Lys-Pro or Ala-Pro using Ang I as substrate: Lys-Pro and Ala-Pro displayed apparent K_i values of 0.5-3 mM and 0.9-5 mM, respectively (Table 1). His-Leu displayed an apparent K_i value of 0.2 mM for the inhibition of Hip-Ala-Pro hydrolysis by wild-type ACE and the C domain and an apparent K_i value of 0.7 mM for the inhibition of hydrolysis by the N domain with enzyme concentration equal to that used for the hydrolysis of Hip-His-Leu (Table 1). These high apparent K_i values of the hydrolysis products indicate a negligible effect on the determination of inhibition constants.

Characterization of Two ACE Synthetic Substrates: Hip-Lys-Pro and Hip-Ala-Pro

To identify a hippuryl-dipeptide substrate more specific for the N domain-active site than the C domain-active site, Hip-Lys-Pro, a novel ACE substrate, and Hip-Ala-Pro were incubated with wild-type ACE and the N or C domain active forms of ACE, and the rate of hydrolysis of the two substrates was calculated from Hip production.

pH dependence and chloride activation of Hip-Lys-Pro and Hip-Ala-Pro hydrolysis. Wild-type ACE and the N and C domains displayed an optimal cleavage of Hip-Lys-Pro at pH 8 (Fig. 2A, inset). In addition, the optimum chloride concentration for the hydrolysis of this substrate was ~20 mM for all three enzymes, with an inhibition of substrate hydrolysis observed at supraoptimal chloride concentrations (>200 mM, Fig. 2A). In the absence of chloride ions, the N domain displayed 80% of optimal activity compared with 10% optimal activity for the C domain.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   pH and chloride dependence of (A) Hip-Lys-Pro and (B) Hip-Ala-Pro hydrolysis by recombinant forms of ACE: ×, wild-type ACE; , C domain; and , N domain. Inset, pH dependence: Hip production (nmol/ml/min) was quantified as described in Experimental Procedures and plotted against pH value. For the chloride dependence of enzyme activity, the velocity (v0) was measured at optimal pH over a range of NaCl concentration (0-600 mM), and the optimal velocity was calculated for each point. The ratio v0/vopt was plotted against the NaCl concentration.

Kinetic parameters of Hip-Lys-Pro and Hip-Ala-Pro hydrolysis. The kinetic parameters of Hip-Lys-Pro hydrolysis were determined under optimal conditions of 20 mM NaCl, pH 8.0. The K_m values calculated from Lineweaver-Burk plots of Hip-Lys-Pro hydrolysis by wild-type ACE and the N and C domains were 112, 74, and 169 µM, respectively, with k_cat values of 167, 36, and 211 sec¹ (mean of three experiments). As shown in Table 2, wild-type ACE and the C domain exhibit a similar k_cat/K_m value, which is 3-fold higher than that for the N domain.

1

Comparison of ACE Inhibitor Potency for the Hydrolysis of Synthetic and Natural Substrates by Wild-Type ACE

The K_i values for the inhibition of Hip-His-Leu or Ang I hydrolysis are similar for each inhibitor but otherwise vary markedly according to the substrate used; for example, lisinopril has a K_i value of 3.9 nM for the inhibition of Hip-Lys-Pro hydrolysis, whereas the same inhibitor displays a K_i value of 0.39 nM with Hip-His-Leu as substrate. Similarly, the K_i value of captopril for the inhibition of AcSDKP hydrolysis is 17-fold lower than that with Hip-Lys-Pro as substrate (Table 3). For the inhibition of physiological substrate hydrolyses, captopril is 16-fold more potent for the inhibition of AcSDKP hydrolysis compared with the inhibition of Ang I hydrolysis. In contrast, lisinopril is equally potent for the inhibition of these two substrate hydrolyses, whereas fosinoprilat is 6-fold more efficient for inhibition of AcSDKP hydrolysis compared with Ang I hydrolysis.

Comparison of ACE Inhibitor Potency for the Hydrolysis of Different Substrates by the ACE N and C Domains

The inhibitory potency of ACE inhibitors toward the ACE N and C domains was determined using synthetic and natural substrates. For wild-type ACE, the potency of the three inhibitors on the N or C domain is related to the substrate used; for example, captopril displays K_i values of 0.1-17 nM (Table 4). Captopril displays a similar potency for the inhibition of the N and C domain-active sites, except for the inhibition of Hip-His-Leu and AcSDKP hydrolysis, for which captopril is 5- and 10-fold more effective, respectively, for inhibition of the N domain than of the C domain. A similar potency of lisinopril for inhibition of the N and C domains is observed toward Hip-Lys-Pro or AcSDKP, whereas lisinopril is 20-fold more effective for C domain inhibition of Hip-His-Leu hydrolysis than for N domain inhibition. For the inhibition of Ang I hydrolysis, captopril inhibits the N and C domains equally efficiently, as does lisinopril; however, fosinoprilat is almost 6-fold more effective at C domain inhibition than at N domain inhibition.

	Discussion

Summary Introduction Procedures Results Discussion References

The tetrapeptide AcSDKP inhibits hematopoietic stem cell proliferation (22); the peptide also appears to block S phase entry of other cell types, such as hepatocytes, lymphocytes, and several continuous cell lines (23, 24). Other inhibitors of hematopoiesis have been identified, such as macrophage inflammatory protein-1, interferon, and tumor necrosis factor; however, the latter two display a complex action, and their hematopoietic effects are likely to be indirect and nonspecific (25). The action of AcSDKP on the inhibition of hematopoietic cell cycling is specific for normal hematopoietic stem cells and has no effect on leukemic cells; thus, AcSDKP was proposed as a therapeutic agent for the protection of normal bone marrow progenitors during chemotherapy (26, 27). In fact, the coadministration of AcSDKP with cytotoxic drugs to mice in vivo increases survival (28).

The hydrolysis of AcSDKP in vitro is inhibited by specific ACE inhibitors, such as captopril and lisinopril (10, 29). Rousseau et al. (10) demonstrated that AcSDKP is hydrolyzed in vitro by ACE and that AcSDKP is a specific substrate of the ACE N domain-active site. Recently, Azizi et al. (11) showed that a single oral dose of captopril (50 mg) administered to healthy subjects induced a 5.5-fold increase in plasma levels of AcSDKP and a 90-99% inhibition of in vitro [³H]AcSDKP hydrolysis. This study demonstrated clearly that AcSDKP is the first natural peptide hydrolyzed specifically by the N domain of ACE not only in vitro but in vivo. The physiological role of the ACE N domain-active site in the degradation of AcSDKP highlights the interest of designing ACE domain-specific inhibitors. Specific inhibition of the ACE N domain could lead to the maintenance of normal undifferentiated stem cells in the quiescent phase during chemotherapy, thus providing protection against cytotoxic drugs while enabling the C domain to perform its established role of ACE in Ang I production. The inhibition of hematopoietic stem cell proliferation in response to the specific ACE inhibitor enalapril has been reported recently (30); these authors showed that the oral administration of enalapril (20 mg/day) over a 15-day period to healthy subjects significantly decreased the granulocyte colony-forming and erythroid burst-forming units, demonstrating the inhibition of hematopoietic stem cell differentiation; this observation was accompanied by a significant increase in undifferentiated cells (granulocyte, erythroid, macrophage, and megakaryocyte colony-forming units). The use of an ACE inhibitor to raise plasma AcSDKP levels is preferable to the administration of exogenous AcSDKP because this peptide requires continuous infusion to be effective due to its constant production and degradation, displaying a half-life in vivo of 4.5 min (31). In contrast, a single oral dose of captopril (50 mg) produces a 5.5-fold longer-lasting increase in plasma AcSDKP (11).

In the current study, we determined the potency of commonly used ACE inhibitors for the hydrolysis of the physiological ACE substrates Ang I and AcSDKP by wild-type ACE and the ACE N and C domains. Such a study could aid the identification or eventual design of ACE inhibitors that exhibit preferential inhibition of the N domain for the hydrolysis of the natural N domain-specific substrate, AcSDKP. In addition, we tested the inhibition of wild-type ACE and the N and C domains using three different synthetic substrates to develop an optimized protocol to allow the large-scale screening of active compounds as an alternative approach for the identification of domain-specific inhibitors.

The synthetic peptide Hip-Lys-Pro was synthesized because it mimics the carboxydipeptide Lys-Pro of AcSDKP and as such could constitute a specific synthetic substrate of the N domain of ACE. However, together with Hip-Ala-Pro, it exhibits a strong affinity for both the N and C domains of ACE, with K_m values close to those of ACE physiological substrates. In addition, both synthetic peptides are hydrolyzed efficiently by all three forms of ACE, with k_cat values comparable to that for Ang I hydrolysis. Therefore, the synthetic substrates used in the current study cannot replace AcSDKP as a specific N domain substrate, but we demonstrate that Hip-Lys-Pro and Hip-Ala-Pro are hydrolyzed efficiently by ACE with kinetic constants in the physiological range. In fact, Hip-Ala-Pro is the best synthetic substrate of this series and could serve as a substitute for Ang I to measure ACE activity; it offers the advantage of being protected against carboxypeptidase hydrolysis by its carboxyl-terminal proline residue. This would be useful in, for example, analysis of ACE activity in tissue samples and biological fluids that contain carboxypeptidases.

The amino acid requirement for a peptide to serve as a substrate for ACE seems to be confined to the residues composing the carboxyl-terminal tripeptide. The presence of a proline residue in P2 (carboxyl-terminal position) increases peptide binding to the different forms of ACE, whereas a histidine residue in P1 (penultimate carboxyl-terminal position) seems to confer a specificity for the C domain. Comparison of Hip-His-Leu and Ang I or of Hip-Lys-Pro and AcSDKP shows that the carboxyl-terminal dipeptide is not sufficient to confer domain substrate specificity. Hip-His-Leu is a relatively specific substrate for the C domain, whereas Ang I is cleaved by both ACE domains. Conversely, Hip-Lys-Pro is hydrolyzed equally well by the two ACE domains, and AcSDKP is a N domain-specific substrate. It is possible that the presence of a benzoyl-glycine moiety (hippuryl) in the P position (antepenultimate carboxyl-terminal position) in place of a phenylalanine residue increases the affinity for the C domain. This hypothesis is supported by the observation that Z-Phe-His-Leu is cleaved almost equally well by the two ACE domains (32). It is possible that the presence of an aspartic acid residue in the P position is imperative for N domain specificity or that the minimum length required for achieving N domain specificity is a tetrapeptide like AcSDKP.

In agreement with previous observations (33), the efficacy of an ACE inhibitor is clearly substrate dependent, with the lowest K_i value observed when each inhibitor was tested with AcSDKP and the highest K_i value observed with Hip-Lys-Pro as substrate. Fosinoprilat is the most potent inhibitor of AcSDKP hydrolysis by the N domain; however, captopril displays the greatest selectivity for the inhibition of AcSDKP and Ang I hydrolysis by wild-type ACE, being 16-fold more potent with AcSDKP as substrate compared with Ang I. This finding is of therapeutic relevance in that captopril is the most efficient ACE inhibitor for blocking AcSDKP metabolism, with a relatively lesser effect on the inhibition of Ang I hydrolysis. These in vitro data will have to be confirmed by in vivo studies in which captopril is administered to enhance plasma AcSDKP with the aim of protecting hematopoietic stem cells during chemotherapy for acute myeloblastic leukemia.¹ Interestingly, for the inhibition of Ang I hydrolysis, captopril is a more potent inhibitor of wild-type ACE compared with the N and C domains. This may be due to the interaction of captopril at one active site of wild-type ACE impeding substrate interaction with the other active site, an effect that is observed with the larger peptide Ang I compared with the other substrates used and only with captopril, which is the weakest competitive inhibitor for wild-type ACE used in this study. Thus, each domain of somatic ACE may not be inhibited independently of the other domain as reported previously (14), but rather a certain degree of cooperativity exists with respect to inhibitor binding.

In conclusion, we demonstrated that the potency of an ACE inhibitor is substrate dependent and that captopril is the most appropriate compound, as identified in this study, for clinical use in the protection of hematopoietic stem cells from cytotoxic drug therapy.