Báo cáo khoa học: Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme docx

Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme Nicolas D. Jullien 1 , Philippe Cuniasse 1 , Dimitris Georgiadis 2 , Athanasios Yiotakis 2 and Vincent Dive 1 1 CEA, De ´ partement d’Inge ´ nerie et d’Etudes des Prote ´ ines, Gif ⁄ Yvette, France 2 Department of Chemistry, Laboratory of Organic Chemistry, University of Athens, Greece Angiotensin-converting enzyme (ACE) in vertebrates is a zinc metallopeptidase involved in the release of angiotensin II and the inactivation of bradykinin, two peptide hormones that play a key role in blood pres- sure regulation and renal and cardiovascular function [1–4]. ACE inhibitors have been on the market for more than 20 years, with successful applications for conditions ranging from mild hypertension to post- myocardial infarction [5,6]. Somatic ACE is a very unusual enzyme which contains two active sites on the same polypeptide chain [7]. Since this discovery, there has been much speculation about the functional significance of the presence of two active sites in the same enzyme [8–13]. At the biochemical level, the pres- ence of two active sites in the same enzyme has hampered the full characterization of substrate and inhibitor selectivity of somatic ACE. To circumvent these limitations, mutants of human ACE containing a single functional active site [14,15] or isolated ACE domains have been utilized to perform these studies [10,11]. Alternative approaches to study directly the properties of both domains in somatic form of ACE are still lacking. The development of the first highly N-domain-specific inhibitor, RXP407 [16], and the recent identification of a C-domain-specific inhibitor, RXPA380 [13], of human ACE may provide such an alternative strategy for studying inhibitor and substrate selectivity of any form of somatic ACE (Scheme 1). To demonstrate its interest, this approach was used to study the selectivity of somatic ACE purified from Keywords active site; angiotensin-converting enzyme (ACE); fluorogenic substrates; phosphinic inhibitors Correspondence V. Dive, CEA, De ´ partement d’Inge ´ nerie et d’Etudes des Prote ´ ines, 91191 Gif ⁄ Yvette Cedex, France Fax: +33 169089071 Tel: +33 169083585 E-mail: vincent.dive@cea.fr (Received 19 December 2005, revised 16 February 2006, accepted 21 February 2006) doi:10.1111/j.1742-4658.2006.05196.x Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild- type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic sub- strate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of func- tional studies performed in animals using N-domain and C-domain sub- strate selectivity data derived only from human ACE. Abbreviations ACE, angiotensin-converting enzyme; DpaOH, N 3 -(2,4-dinitrophenyl)-L-2,3-diaminopropionyl); Mca, (7-methoxycoumarin-4-yl)acetyl. 1772 FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS mouse and rat, as compared with human recombinant somatic ACE, in cleaving the Mca-Ala-Ser-Asp-Lys- DpaOH substrate (Mca-Ala), as well as a new fluorogenic substrate of this enzyme, Mca-Arg-Pro- Pro-Gly-Phe-Ser-Pro-DpaOH (Mca-BK (1)8) ). A few years ago, we reported that there are slight variations in the potency of RXP407 toward the N-domain of human, mouse and rat [17]. The present study extends our previous observations by showing unexpected dif- ferences between the N-domain and C-domain sub- strate selectivity of these three ACE species. Models of the N-domain and C-domain of these ACE enzymes were developed based on the crystal structure of germi- nal human ACE [18]. From these, the differences in substrate selectivity between the human, rat and mouse enzymes can tentatively be explained by the presence of discrete amino-acid substitutions in the active site. Results Titration of somatic ACE by RXP407 using Mca-Ala substrate The profile of inhibition of human somatic ACE by RXP407, using the Mca-Ala substrate, typically exhib- its two asymmetrical parts (Fig. 1A, closed circle). As discussed in previous papers [16,19], each part reflects first the binding of RXP407 to the N-domain at low inhibitor concentration, followed at higher concentra- tions by inhibitor binding to the C-domain. The loca- tion of the inflection points in this profile provides a direct measure of the IC 50 value (concentration that causes 50% inhibition) for both domains. In addition, the value of the inhibition percentage, determined after inhibitor binding to the active site showing the highest affinity, was demonstrated to depend on the selectivity with which the substrate is processed by the N-domain and C-domain (indicated by the arrow in Fig. 1A). Thus, information on the selectivity of both the sub- strate and the inhibitor for the N-domain and C-domain can be determined from such profiles. Remarkably, with the same substrate, RXP407 inhi- bition profiles of somatic ACE purified from mouse (Fig. 1A, open circle) and rats (closed square) are quite different from that observed for human ACE. For mouse ACE, the shape of the inhibition profile sug- gests the titration of a single active site to which RXP407 binds with high affinity. Such a profile can be expected for a substrate that is mostly cleaved by only one active site, probably the N-domain given the RXP407 selectivity. In contrast with mouse and human ACE forms, titration of rat ACE by RXP407 yields a profile that is shifted to the right. The shape of this profile is consistent with the binding of RXP407 to the N-domain, at low concentration, and to the C-domain at higher concentration. The differences observed between the human and rat profiles are best explained by the fact that the affinity of RXP407 for the rat N-domain is lower than that for the human N-domain, and Mca-Ala selectivity differs between rat and human ACE. In the human enzyme, the binding of RXP407 to the N-domain led to 80% inhibition of its activity (arrow in Fig. 1A, closed circle), whereas in the rat enzyme binding of RXP407 to the N-domain only gave 20% inhibition (closed square, Fig. 1A). The second part of the inhibition profile, from 20% to 100%, observed for rat ACE represents RXP407 binding to the C-domain and highlights that this substrate is more efficiently cleaved by the C-domain of rat ACE. The above experiments suggest that the selectivity by which the Mca-Ala substrate is cleaved by the N-domain and C-domain of somatic ACE depends on the ACE species. To support this hypothesis, mouse and rat ACE forms were titrated with the RXPA380, an ACE inhibitor previously shown to exhibit high selectivity for the C-domain of somatic human ACE. Titration of somatic ACE by RXPA380 using Mca-Ala substrate The titration of human ACE by RXPA380, using Mca-Ala substrate (Fig. 1B, closed circles), gave a pro- file that differs from that obtained by RXP407 titra- tion (Fig. 1A, closed circles). Binding of RXPA380 to the human ACE C-domain only reduced the enzyme activity by 20% (left part of the profile). This observa- P O - H N CH 2 H N O CH 3 NH 2 O O N H COO - O H 3 C O H 3 C RXP407 P O H N CH 2 C H N OCH 2 OH O HN O O OH RXPA380 Scheme 1. N. D. Jullien et al. ACE substrate specificity FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS 1773 tion is in agreement with the suggestion made above that Mca-Ala is more efficiently cleaved by the human ACE N-domain. In fact, titration of the N-domain using higher RXPA380 concentration (right part of the profile) raised the inhibition from 20% to 100%. In rat, on the other hand, ACE binding of RXPA380 to the C-domain reduced the enzyme activ- ity by 85% (closed square, Fig. 1B), as compared with the 20% inhibition observed with human ACE. Thus, titration of rat ACE by both RXP compounds led to a similar proposal, the higher catalytic efficiency of the C-domain in cleaving the Mca-Ala substrate. Titration of the mouse ACE C-domain using low concentrations of RXPA380 (Fig. 1B, open circle) did not affect enzyme activity. Inhibition of enzyme activ- ity only occurred when RXPA380 started to bind to the N-domain of ACE, at very high concentration. This observation is in agreement with the RXP407 inhibition profile of mouse ACE, suggesting that the Mca-Ala substrate is mostly cleaved by the N-domain of mouse ACE. To conclude this part, the inhibition profiles obtained with both RXP inhibitors confirm that the catalytic efficiency by which Mca-Ala is processed by the N-domain and C-domain varies according to the ACE species. For rat and human enzymes, estimation of IC 50 values for the binding of the inhibitor to the N-domain and C-domain can be deduced from the inhibition profiles established with the Mca-Ala sub- strate. For mouse ACE, as this substrate is mostly cleaved by the N-domain, only IC 50 values for inhib- itor binding to the N-domain can be obtained from the inhibition profile. Thus, affinity of the inhibitor for the mouse ACE C-domain cannot be determined using this substrate. This limitation has been overcome by developing another fluorogenic substrate of ACE. Titration of somatic ACE with RXP407 and RXPA380 using Mca-BK (1)8) substrate The presence of two distinct parts in all inhibition pro- files (Fig. 1C,D) clearly indicates that the Mca-BK (1)8) AB D C Fig. 1. Inhibition profiles (% inhibition) of human (d), mouse (s) and rat (n) somatic ACE with RXP407 (A) or RXPA380 (B) when the substrate used was Mca-Ala-Ser-Asp-Lys-DpaOH (Mca-Ala, 8 l M). Arrows show the inhibition of human and rat N-domain by RXP407 (A) or human and rat C-domain by RXPA380 (B). Inhibition profiles of the three ACE forms with RXP407 (C) or RXPA380 (D) when the substrate was Mca-Arg- Pro-Pro-Gly-Phe-Ser-Pro-DpaOH [Mca-BK (1)8) ,5lM]. Arrows show the inhibition of human, mouse and rat N-domain by RXP407 (C) or human, mouse and rat C-domain by RXPA380 (D). All the assays were carried out in 50 m M Hepes buffer (pH 6.8) ⁄ 200 mM NaCl, at 25 °C. Continu- ous lines display the simulated profiles obtained with the DYNAFIT program that represent the best fit of the experimental data. ACE substrate specificity N. D. Jullien et al. 1774 FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS substrate is cleaved by the two ACE active sites. The higher levels of inhibition observed after binding of RXPA380 to the ACE C-domains (Fig. 1D), as com- pared with the inhibition levels reached by RXP407 binding to the N-domain (Fig. 1C), suggest that Mca- BK (1)8) displays C-domain selectivity, the degree of which depends on the ACE species. Titration of the ACE C-domain with RXPA380 promoted higher ACE inhibition (55% to 90% inhibition, depending on the ACE species, Fig. 1D) than blockade of the N-domain by RXP407 (10% to 45% inhibition, depending on the ACE species, Fig. 1C). Comparison of the profiles in Fig. 1C,D shows that Mca-BK (1)8) displays the highest C-domain selectivity toward rat ACE. In contrast with Mca-Ala, IC 50 values for the binding of the RXP com- pounds to both the N-domain and C-domain of mouse ACE can be estimated using the Mca-BK (1)8) sub- strate. From the similarities between the different profiles, it can be concluded that the affinity and selec- tivity of both inhibitors for the N-domain and C-domain of different ACE species are conserved, except for RXP407 affinity for the N-domain of rat ACE (see below). Catalytic efficiencies of the ACE N-domain and C-domain in cleaving the Mca-Ala and Mca-BK (1)8) substrates The inhibition profiles described above indicate the approximate inhibitor concentration required to block only the N-domain or C-domain of the different ACE species. Specific blockade of only one ACE active site by RXP compounds may be a way to determine the catalytic properties of the second ACE active site, free of inhibitor, assuming that the two active sites function independently. To check this proposal, the catalytic parameters K m and k cat of the different ACE species were determined in the absence or presence of one RXP compound. In these experiments, the concentra- tion of each RXP compound was chosen in order to inhibit mostly just one ACE active site. The results reported in Table 1 suggest that, within experimental error, the catalytic efficiencies of the N-domain and C-domain in cleaving the Mca-Ala substrate, in the presence of RXP compounds, are in good agreement with the catalytic efficiency determined for the enzyme without inhibitor. Specifically, for each ACE species, the catalytic efficiency of the free enzyme corresponds to the sum of the catalytic efficiencies determined for the N-domain and C-domain in the presence of inhib- itor. These results lend credence to the hypothesis that, for this substrate, each ACE active site functions inde- pendently. Moreover, these data confirm that each ACE species processes this substrate with a particular selectivity. From these experiments, the Mca-Ala sub- strate turns out to be an N-domain-selective substrate of mouse ACE, showing the highest catalytic efficiency in cleaving this substrate, as compared with human and rat N-domain. The two active sites of human ACE hydrolyze this substrate, but the N-domain is more efficient than the C-domain in catalyzing this reaction. The reverse is observed for rat ACE, this substrate being better cleaved by the C-domain. Inter- estingly, although significant differences between the catalytic efficiency of either the N-domain or C-domain are observed between these ACE species, the overall activity of both domains is constant for the three species. Thus, in this case, the gene duplication of ACE may be a way to keep the catalytic efficiency of the somatic enzyme intact, while allowing variations in the N-domain and C-domain catalytic properties. Overall, these data on interspecies Mca-Ala substrate Table 1. Kinetic parameters for the hydrolysis of the Mca-Ala substrate by rat, mouse and human somatic ACE. Kinetic parameters k cat and K m were obtained by inhibiting the activity of the C-domain of ACE with RXPA380 (active N-domain) or by inhibiting the N-domain activity with RXP407 (active C-domain)(see Experimental procedures). N + C is the sum of the k cat ⁄ K m values reported for the N-domain and C-domain. k cat and K m were obtained by the direct linear plot method; the confidence limits are indicated in parentheses. k cat ⁄ K m values were calculated using the experimental k cat and K m values. ND, not determined, no detectable activity. Parameter Rat Mouse Human N-domain k cat (s )1 ) 2.8 (2.6–3.0) 13.8 (12.8–15.2) 14.0 (12.9–14.8) K m (lM) 45.9 (37.8–52.9) 26.1 (19.9–33.6) 35.7 (26.4–40.8) k cat ⁄ K m (s )1 ÆlM) 0.06 0.53 0.39 C-domain k cat (s )1 ) 19.1 (17.9–20.0) ND 4.2 (37.–4.7) K m (lM) 49.5 (43.1–53.3) ND 64.6 (52.4–77.1) k cat ⁄ K m (s )1 ÆlM) 0.39 ND 0.06 N+C k cat ⁄ K m (s )1 ÆlM) 0.45 – 0.45 Somatic ACE k cat (s )1 ) 24.7 (22.5–26.3) 14.2 (13.6–15.8) 16.5 (15.1–17.7) K m (lM) 46.9 (38.4–52.5) 24.6 (17.9–30.8) 34.8 (27.2–40.9) k cat ⁄ K m (s )1 ÆlM) 0.53 0.57 0.47 N. D. Jullien et al. ACE substrate specificity FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS 1775 selectivity confirm our interpretation of the inhibition profiles reported in Fig. 1. Similar experiments were performed using the Mca- BK (1)8) substrate (Table 2). For this substrate, the activity of the free enzyme also corresponds to the sum of the activities determined for each domain in the presence of an RXP compound. This substrate was cleaved by the two ACE active sites of different spe- cies, the selectivity of cleavage varying with the source of ACE. In particular, this substrate exhibits a marked C-domain selectivity toward rat ACE, whereas it dis- plays only a very slight C-domain selectivity when tes- ted with mouse ACE. In addition, this substrate is cleaved 10 times faster by rat ACE than mouse ACE. As compared with Mca-Ala, the activity of the somatic enzyme varies between the three ACE species. Determination of RXP407 and RXPA380 K i values for ACE species Inhibition profiles as reported in Fig. 1 can be used to determine the K i values of the RXP compounds for the N-domain and C-domain of each ACE species. Assuming that each ACE active site functions inde- pendently, we previously showed that simulated inhibi- tion profiles that best reproduce the experimental data can provide access to K i values [16,19]. Such simula- tions rely on the use of inputs, notably the catalytic parameters displayed by each ACE domain in cleaving the substrate used in the experiments. The results reported above, in the presence of RXP inhibitor blocking only one active site, provide approximate val- ues of the catalytic parameters displayed by each ACE domain for cleaving the Mca-Ala and Mca-BK (1)8) substrates. These values were thus tentatively used to simulate inhibition profiles able to reproduce the experimental inhibition profiles. As shown in Fig. 1, excellent fits between experimental data and simula- ted curves were generally observed. The K i values as determined by this approach (Table 3) indicate that RXP407 and RXPA380 are, respectively, highly N-domain-selective and C-domain-selective inhibitors of the three ACE species, whatever the substrate used. For human ACE, the K i values determined by this fitting approach were in close agreement with those previously reported, using mutants of this enzyme con- taining only one functional active site [16]. With both substrates, RXP407 appears slightly less potent toward the N-domain of rat ACE, whereas RXPA380 displays similar affinity toward the C-domain of each ACE spe- cies, showing three orders of magnitude in selectivity. Inhibitor and substrate selectivity relationships with the ACE sequences In a previous paper, we proposed active-site residues contributing to inhibitor selectivity based on a model of Table 2. Kinetic parameters for the hydrolysis of the Mca-BK (1)8) substrate by rat, mouse and human somatic ACE. Kinetic parameters k cat and K m were obtained by inhibiting the activity of the C-domain of ACE with RXPA380 (active N-domain) or by inhibiting the N-domain activ- ity with RXP407 (active C-domain)(see Experimental procedures). N + C is the sum of the k cat ⁄ K m values reported for the N-domain and C-domain. k cat and K m were obtained by the direct linear plot method; the confidence limits are indicated in parentheses. k cat ⁄ K m values were calculated using the experimental k cat and K m values. Parameter Rat Mouse Human N-domain k cat (s )1 ) 38.4 (28.9–58.6) 8.9 (7.9–10.5) 17.8 (16.6–20.3) K m (lM) 58.0 (22.9–65.6) 30 (18.8–36.4) 26.1 (18.9–31.3) k cat ⁄ K m (s )1 ÆlM) 0.66 0.30 0.68 C-domain k cat (s )1 ) 110.9 (105.6–119.3) 4.1 (3.7–4.5) 15.7 (15.1–16.0) K m (lM) 18.9 (14.8–21.3) 12.1 (8.5–14.2) 9.2 (8.3–10.3) k cat ⁄ K m (s )1 ÆlM) 5.87 0.34 1.71 N+C k cat ⁄ K m (s )1 ÆlM) 6.53 0.64 2.39 Somatic ACE k cat (s )1 ) 119.0 (108.7–133.3) 12.4 (11.3–14.3) 38.8 (37.3–40.0) K m (lM) 20.2 (14.2–25.4) 21.0 (13.1–24.2) 13.9 (12.5–14.9) k cat ⁄ K m (s )1 ÆlM) 5.89 0.59 2.79 Table 3. Potency and selectivity of RXP407 and RXPA380 toward the N-domain and C-domain of human, mouse and rat somatic ACE. K i(app) values were determined from the simulations that best reproduced the inhibition profiles reported in Fig. 1. ND, not deter- mined, no detectable activity. Domain K i(app) values (nM) Mca-Ala Mca-BK (1)8) Rat Mouse Human Rat Mouse Human RXP 407 N 55 8 10 30 13 16 C 8500 ND 4000 10000 6000 8000 RXP A380 N 7000 3500 5500 10000 5500 5500 C 2.6 ND 5 7 8 10 ACE substrate specificity N. D. Jullien et al. 1776 FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS RXPA380 in complex with human ACE [20]. In partic- ular, two bulky and hydrophobic residues of the human ACE C-domain, Val955 and Val956 located in the S 2 ¢ subsite, were proposed to provide favorable interactions with the tryptophan side chain of RXPA380. These interactions should be lost in the N-domain, as valine residues are replaced by smaller polar residues (Ser357 and Thr358). These differences were suggested to con- tribute to RXPA380 selectivity. As depicted in Table 4, rat and mouse ACE C-domain exhibit bulky hydropho- bic residues at positions 955 and 956 and smaller polar residues at positions 357 and 358 in the N-domain. This conservation in the S 2 ¢ binding pocket of ACE fits with the similar potency and selectivity displayed by RXPA380 towards the different ACE species. Follow- ing the same strategy, a model of RXP407 interaction with the human ACE N-domain has been developed to identify residues of the S 2 ,S 1 ,S 1 ¢ and S 2 ¢ pockets that may influence either inhibitor or substrate selectivity. This model reveals (Fig. 2) that, among the residues defining the S 2 ,S 1 ,S 1 ¢ and S 2 ¢ pockets, two residues may greatly influence the RXP407 potency and selectiv- ity. In fact, in the N-domain, the aspartyl side chain of RXP407 is observed to interact with Tyr369 and Arg381 through hydrogen-bond contacts. Indeed, short distances between the O d1 and O d2 atoms of the RXP407 aspartyl residue and Tyr369 ⁄ O g atom on one side and Arg381 ⁄ N f ,N g atoms on the other side are observed in this model (Fig. 2). Similar interactions cannot take place in the C-domain, as these two resi- dues are replaced by Phe967 and Glu979. As the aspar- tyl residue in RXP407 is the key residue controlling inhibitor selectivity, we suggest that the mutations observed in the 369 ⁄ 967 and 381 ⁄ 979 positions may contribute to RXP407 selectivity. Rat and mouse ACE display the same feature as is observed in human ACE, which is consistent with the potency and selectivity dis- played by RXP407 toward the rat and mouse enzymes. It should be mentioned that a model of RXP407 inter- action with human N-domain was previously reported that differs from our model [6]. In that model, the Table 4. Residues located in the S 2 ,S 1 ,S 1 ¢ and S 2 ¢ subsites of ACE enzymes possibly implicated in the substrate selectivity. Residues that change between ACE species are in bold and those that change between the N-domain and C-domain are in italics. N-domain C-domain Rat Mouse Human Rat Mouse Human S 2 Tyr369 Tyr369 Tyr369 Phe967 Phe967 Phe967 Arg381 Arg381 Arg381 Glu979 Glu979 Glu979 S 1 Ser39 Ser39 Ser39 Asn642 Asn642 Asn642 Ser119 Ser119 Ser119 Glu719 Glu719 Glu719 Ser494 Asn494 Asn494 Ala1092 Ala1092 Ser1092 Val495 Val495 Val495 Asn1093 Asn1093 Ser1093 Thr496 Thr496 Thr496 Val1094 Val1094 Val1094 S 1 ¢ Ala332 Ala332 Ala332 Ala930 Pro930 Ala930 S 2 ¢ Ser260 Ser260 Ser260 Thr858 Thr858 Thr858 Asp354 Glu354 Asp354 Glu952 Glu952 Glu952 Ser357 Ala357 Ser357 Val955 Val955 Val955 Thr358 Thr358 Thr358 Ile956 Ile956 Val956 Glu431 Glu431 Glu431 Asp1029 Asp1029 Asp1029 Fig. 2. Detail of the human ACE N-domain active in the interaction with RXP407. The active-site helix carrying the HEXXH sequence is colored yellow. RXP407 is colored by atom type; the carbon atoms are in green. Active-site residues located at a distance less than 5A ˚ from RXP407 are in light blue when they are observed to change between ACE species (in either the N-domain or C-domain). Three residues interacting with RXP407 in the model are displayed in orange; these residues are conserved between the N-domain of the three ACE species (N-domain numbering). Distances in ang- stroms between atoms of Arg381, Tyr369 and the aspartate atoms of RXP407 are reported. Hydrogens and zinc atom are omitted. The figure was prepared with PYMOL software. N. D. Jullien et al. ACE substrate specificity FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS 1777 Tyr369 and Arg381 residues were observed to interact with the acetyl group of RXP407. As mentioned above, our model seems to better explain the key role of the aspartyl residue in inhibitor selectivity. As shown in Fig. 2 (see also Table 4), the model of RXP407 interac- tion with the N-domain reveals that several positions in the ACE active site, covering the S 1 to S 2 ¢ subsites, change between the three ACE species, in either the N-domain or C-domain (494, 354, 357, 930, 956, 1092 and 1093). These mutations may contribute to the dif- ferences in Mca-Ala and Mca-BK (1)8) selectivity observed with the three ACE species. As the major dif- ferences in catalytic properties between the N-domain and C-domain of the three ACE species is reflected by changes in k cat but not K m , it might be suggested that all the aforementioned residues participate in the stabil- ization of the transition state. Discussion The use of highly selective inhibitors of ACE makes it possible to inhibit one enzyme active site and derive catalytic parameters for the other active site, free of inhibitor. The good agreement between the catalytic parameters obtained by blocking one ACE active site and those determined with the free somatic form using inhibitor profiles (Tables 1 and 2) validates the use of selective inhibitors as convenient tools to study the spe- cificity of the somatic form of ACE. Thus, with this approach, the catalytic property of each domain towards the hydrolysis of a substrate can be determined without the need to produce mutants or isolate ACE domains. The results of this approach are also consis- tent with the shape of the inhibition profiles observed for each ACE species. Intuitive interpretation of the inhibition profiles of mouse and rat somatic ACE with RXP407 and RXPA380 (Fig. 1A,B) suggests that the Mca-Ala substrate is a strict N-domain-selective sub- strate of mouse ACE, whereas it is almost cleaved by the C-domain of rat ACE. This interpretation is con- firmed by the kinetic parameters reported in Table 1. Overall, this work based on the study of two syn- thetic substrates and three different ACE species reveals that the N-domain and C-domain substrate selectivity is not conserved between different ACE spe- cies, implying that the functional role played by each domain may change from one species to another. Thus, any conclusion on the N-domain and C-domain sub- strate selectivity based on a single ACE species could be misleading. In this respect, it is worth mentioning that the N-domain and C-domain selectivity towards physiological substrates of ACE (angiotensin I, brady- kinin and N-acetyl-seryl-aspartyl-lysyl-proline) has been established only for the human enzyme [21], because of the availability of a mutant form of the human enzyme containing a single functional active site. As far as sub- strate selectivity is concerned, data obtained in animal models could be misinterpreted if the properties of the human enzyme are used. The approach presented in this study, which determines the selectivity of any sub- strate whatever the source of ACE, could be used to resolve this important issue. The study of mouse and rat ACE selectivity toward natural substrates, such as N-acetyl-seryl-aspartyl-lysyl-proline, angiotensin I and bradykinin, should be possible using the same approach, as the potency and selectivity of the RXP inhibitors did not appear to vary to any great extent toward these ACE enzymes. Such studies are relevant in the light of the differences exhibited by rat and mouse for the hydrolysis of Mca-Ala and Mca-BK, which can be viewed as mimics of he natural N-acetyl- seryl-aspartyl-lysyl-proline and BK 1-8 peptides. It should be noted that, until the development of inhibi- tors able to block specifically only one ACE active site, the question of ACE domain selectivity was not so crit- ical. The correct interpretation of in vivo data obtained after animal treatment with RXP inhibitors requires a good knowledge of the specificity of the domain, free of inhibitor, towards the physiological substrates of ACE. This requirement also applies to the natural an- giotensin-(1–7) peptide, which was reported to act as a C-domain selective inhibitor of ACE [11]. This know- ledge is essential to appreciate whether ACE domain- specific inhibitors represent a new class of inhibitors showing particular pharmacological profiles of medical interest. Another major concern is that, according to the data reported in this study, evaluation of these domain-selective ACE inhibitors may vary between rat and mouse models, rendering extrapolation of these results to the human situation problematic. In support of our experimental results highlighting differences in N-domain and C-domain substrate selec- tivity between ACE species, our nonexhaustive compar- ison of mouse, rat and human ACE sequences around the active site of these enzymes identifies several resi- dues that change (Table 4). In human ACE, it has already been reported that Asn494 occurs in an N-gly- cosylation sequon (NTV), that is unique to the N- domain [6]. Any glycosylation of this residue, which is located in the active site, is expected to greatly influence enzyme activity. Interestingly, this asparagine is replaced by serine in the rat N-domain. Whether this mutation results in no glycosylation or O-glycosylation of Ser494 in rat ACE is not known [22], but, in any case, it can be expected to affect enzyme activity. The low efficiency of the rat N-domain in cleaving the ACE substrate specificity N. D. Jullien et al. 1778 FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS Mca-Ala substrate, compared with the N-domain of the mouse and human enzymes, may in part be due to this mutation. The resolution of the 3D structure of the germinal form of ACE has provided a strong impetus for further studies aimed at understanding, at the molecular level, how the two active sites of somatic ACE function [18]. The data reported in this study pro- vide supplementary information about the residues that should be considered in any ACE models intended to explain the selectivity of this enzyme. These residues should be considered in future mutagenesis studies designed to map the residues involved in the specificity of both the N-domain and C-domain of ACE. Gene duplication leading to the presence of several protease domains within a single polypeptide chain is a rare event, so far only observed in ACE, carboxypeptidase D and polyserases [23,24]. The putative functional advantages that may result from such complex protein assembly remain elusive. Rat and mouse ACE were observed to cleave the Mca-Ala peptide with similar efficiency, but surprisingly the contribution of each domain to this cleavage varies considerably in these ACE species. In this case, independent evolution of each domain does not affect the global enzyme activity. Beside complementary enzyme activity, many other functional advantages have been proposed for these multidomain proteases, such as positive or negative co- operativity [23–26]. Clearly, a better understanding of the functional role played by each domain in these intriguing proteases will require additional studies. Experimental procedures Chemicals RXP407 and RXPA380 (Scheme 1) were synthesized as des- cribed previously in [16] and [13], respectively. Quinaprilat was kindly provided by Professor P. Corvol of the Institut National de la Sante ´ et de la Recherche Me ´ dicale, Unite ´ 36, Paris, France. Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH (Mca-BK (1)8) ) substrate [Mca, (7-methoxycoumarin-4- yl)acetyl; DpaOH, N 3 -(2,4-dinitrophenyl)-l-2,3-diaminopro- pionyl)] was prepared by following the procedure described for Mca-Ala-Ser-Asp-Lys-DpaOH (Mca-Ala) [16]. Enzymes Human wild-type somatic ACE was obtained by stable expression in Chinese hamster ovary cells transfected with appropriate ACE cDNA [14]. This material was kindly pro- vided by P. Corvol (Colle ` ge de France, Paris, France). Expression and purification of ACE were performed as pre- viously described [14]. Mouse and rat somatic ACE were purified by affinity chromatography as described previously [17], from lung homogenates obtained from C57BL ⁄ 6 mice and Lewis rats (Charles River France, L’arbresle, France). ACEs purified by this method appeared as homogeneous single bands on SDS ⁄ PAGE. Enzyme assays Continuous assays were performed by recording the fluores- cence increase at 405 nm (e ex ¼ 320 nm) induced by the cleavage of Mca-Ala and Mca-BK (1)8) substrates by ACE, using black, flat-bottomed, 96-well nonbinding surface plates (Corning-Costar, Schiphol-Rijk, the Netherlands). Assays were carried out in triplicate, at 25 °C, in 50 mm Hepes (pH 6.8) ⁄ 200 mm NaCl. Fluorescence signals were monitored using a Fluoroscan Ascent photon counter spec- trophotometer (Thermo-Labsystems, Courtaboeuf, France) equipped with a temperature control device and a plate sha- ker. The substrate and enzyme concentrations for the experiments were chosen so as to remain well below 10% of substrate utilization and to observe initial rates. Concen- trations of substrate solutions were determined spectropho- tometrically using e 328nm ¼ 12 900 m )1 Æcm )1 . The site of cleavage of Mca-BK (1)8) by ACE enzymes was determined by HPLC analysis coupled with MS. For the three ACE species, a unique cleavage was observed at Phe-Ser. Determination of kinetic parameters The apparent kinetic parameters K m and k cat for the hydro- lysis of the substrates Mca-Ala and Mca-BK (1)8) by the N-domain or the C-domain of human, mouse, or rat ACE were determined by blocking the activity of the C-domain by 150 nm RXPA380 or the activity of the N-domain by 350 nm RXP407, except for rat ACE for which the N-domain was inhibited using 750 nm RXP407. At these concentrations, RXP compounds mainly inhibit the activity of one active site, allowing the determination of the kinetic parameters of the other active site, free of inhibitor. For each species and each inhibitor, ACE was incubated with the inhibitor for 45 min before substrate addition. The kin- etic parameters were determined using the direct linear plot method [27–30] and substrate concentration ranges of 10– 122 lm for Mca-Ala and 2–71 lm for Mca-BK 1-8 . Concen- trations of ACE in these experiments were determined by titration of the enzyme with quinaprilat. Inhibition studies For inhibition studies, all inhibitors were preincubated for 45 min before initiation of the reaction by substrate addition. The substrate concentration used was 8 lm for Mca-Ala and 5 lm for Mca-BK (1)8) . Data were collected every 30 s over a period of 25 min. Inhibitor concentrations were selected in N. D. Jullien et al. ACE substrate specificity FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS 1779 order to observe a full range of inhibition percentages. With the wild-type somatic ACE from the three species (human, mouse, rat), K i(app) values were estimated from simulations. Inhibition profiles of ACE by the different inhibitors with Mca-Ala and Mca-BK (1)8) substrates were simulated using the program dynafit from P. Kuzmic [31], as described in [16,19]. A two-active-site model was used to fit these profiles, except for the titration profiles of mouse ACE using the Mca-Ala substrate, for which a one-active-site model was used. In some cases, in order to achieve a better fit between simulated and experimental data, k catN ⁄ C values were varied within the limit of the experimental confidence values of these parameters (Tables 1 and 2), keeping the selectivity (k cat ⁄ K m ) N ⁄ (k cat ⁄ K m ) C ratio constant. Identification of the residues covering the S 2, S 1, S 1 ¢ and S 2 ¢ binding sites in ACE A model of RXP407 interaction with the human ACE N- domain has been developed to identify residues of the S 2 , S 1 ,S 1 ¢ and S 2 ¢ pockets that could influence either inhibitor or substrate selectivity in this enzyme. The conservation of these residues was then checked, using aligned sequences of the N-domain and C-domain of human, mouse and rat enzymes. The model of the N-domain of human ACE (ACE-N) was achieved by homology modeling using the 3D structure of the human germinal form of this enzyme reported in complex with lizinopril (pdbcode 1086) [18]. The initial structure of the N-domain of ACE was obtained with the program modeller 6v2 [32] by alignment with the germinal form of ACE (55.2% identity in 583 amino acids overlap). The resulting structure was then used to build an initial structure of the complex N-domain–RXP407. The structure of RXP407 was constructed with the insight ii software (Accelrys Inc., Sandiego, CA, USA). To obtain the initial structure of the N-domain–RXP407 complex, we used the previously reported model of C-domain–RXPA380 [20]. The N-domain structure was superimposed on the C-domain structure and the RXP407 inhibitor was superim- posed on RXPA380 by minimizing the r.m.s.d. on the posi- tion of the common backbone atoms. The resulting N-domain–RXP407 structure was then refined using a pro- tocol of molecular dynamics with the program charmm (v.27) [33]. The charmm force field version 22 was used [34]. Geometrical and nonbonded parameters for the phos- phinic inhibitor RXP407 were derived from ab initio quan- tum calculations with the program gaussian98 (Gaussian Inc., Pittsburgh, PA, USA). These calculations were per- formed at the MP2 level of theory using a 6–31 + G(d,p) basis set. To preserve the structure of the protein during the relaxation of the complexes, harmonic restraints were applied to the atomic position of several sets of atoms. The harmonic constants were set to 100, 5 and 0.5 kcalÆ mol )1 ÆA ˚ )2 for the ions and their chelating residues, the atoms of the protein situated at a distance greater than 5 A ˚ of the inhibitor, and the backbone and Cb atoms of the inhibitor and those of the protein located at a distance smaller than 5 A ˚ of the inhibitor, respectively. No har- monic restraints were applied to hydrogen atoms. The ini- tial step of the relaxation protocol consists of an initial 2000 cycles of Adopted Basis Newton-Raphson energy min- imization. Then 100 000 steps of molecular dynamics using the Verlet algorithm were undertaken. The integration step was set to 0.0004 ps. The temperature was gradually increased by 25 K each 1000 steps to reach 300 K. This molecular dynamics was followed by 5000 cycles of energy minimization. During the calculations, the nonbonded interactions were modeled using a Lennard-Jones function and a coulombic electrostatic term with a nonbonded cut- off of 16 A ˚ . The dielectric constant was set to 1. The result- ing structure was then analyzed with the program pymol (Delano Scientific Inc., San Francisco, CA, USA). Acknowledgements This work was supported by the CEA ( Commissariat a ` l’Energie Atomique). N.J. was funded through a fellow- ship from French Government and Servier Institut. References 1 Erdos EG (1990) Angiotensin I converting enzyme and the changes in our concepts through the years. Lewis K. Dahl memorial lecture. Hypertension 16, 363–370. 2 Bhoola KD, Figueroa CD & Worthy K (1992) Bioregu- lation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev 44, 1–80. 3 Gavras H (1994) Corcoran Lecture. Angiotensin-con- verting enzyme inhibition and the heart. Hypertension 23, 813–818. 4 Dzau VJ (2001) Theodore Cooper Lecture: tissue angio- tensin and pathobiology of vascular disease: a unifying hypothesis. Hypertension 37, 1047–1052. 5 Zaman MA, Oparil S & Calhoun DA (2002) Drugs tar- geting the renin-angiotensin-aldosterone system. Nat Rev Drug Discov 1, 621–636. 6 Acharya KR, Sturrock ED, Riordan JF & Ehlers MR (2003) ACE revisited: a new target for structure-based drug design. Nat Rev Drug Discov 2, 891–902. 7 Soubrier F, Alhenc-Gelas F, Hubert C, Allegrini J, John M, Tregear G & Corvol P (1988) Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning. Proc Natl Acad Sci USA 85, 9386–9390. 8 Rousseau A, Michaud A, Chauvet MT, Lenfant M & Corvol P (1995) The hemoregulatory peptide N-acetyl- Ser-Asp-Lys-Pro is a natural and specific substrate of the N-terminal active site of human angiotensin-convert- ing enzyme. J Biol Chem 270, 3656–3661. ACE substrate specificity N. D. Jullien et al. 1780 FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS 9 Deddish PA, Wang LX, Jackman HL, Michel B, Wang J, Skidgel RA & Erdos EG (1996) Single-domain angioten- sin I converting enzyme (kininase II): characterization and properties. J Pharmacol Exp Ther 279, 1582–1589. 10 Deddish PA, Jackman HL, Skidgel RA & Erdos EG (1997) Differences in the hydrolysis of enkephalin congeners by the two domains of angiotensin converting enzyme. Biochem Pharmacol 53, 1459–1463. 11 Deddish PA, Marcic B, Jackman HL, Wang HZ, Skid- gel RA & Erdos EG (1998) N-domain-specific substrate and C-domain inhibitors of angiotensin-converting enzyme: angiotensin-(1–7) and keto-ACE. Hypertension 31, 912–917. 12 Junot C et al. (2001) RXP 407, a selective inhibitor of the N-domain of angiotensin I-converting enzyme, blocks in vivo the degradation of hemoregulatory pep- tide acetyl-Ser-Asp-Lys-Pro with no effect on angioten- sin I hydrolysis. J Pharmacol Exp Ther 297, 606–611. 13 Georgiadis D, Beau F, Czarny B, Cotton J, Yiotakis A & Dive V (2003) Roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angio- tensin I and bradykinin: insights from selective inhibi- tors. Circ Res 93, 148–154. 14 Wei L, Alhenc-Gelas F, Corvol P & Clauser E (1991) The two homologous domains of human angiotensin I-converting enzyme are both catalytically active. J Biol Chem 266, 9002–9008. 15 Jaspard E, Wei L & Alhenc-Gelas F (1993) Differences in the properties and enzymatic specificities of the two active sites of angiotensin I-converting enzyme (kininase II). Studies with bradykinin and other natural peptides. J Biol Chem 268, 9496–9503. 16 Dive V, Cotton J, Yiotakis A, Michaud A, Vassiliou S, Jiracek J, Vazeux G, Chauvet MT, Cuniasse P & Corvol P (1999) RXP 407, a phosphinic peptide, is a potent inhibitor of angiotensin I converting enzyme able to dif- ferentiate between its two active sites. Proc Natl Acad Sci USA 96, 4330–4335. 17 Vazeux G, Cotton J, Cuniasse P & Dive V (2001) Potency and selectivity of RXP407 on human, rat, and mouse angiotensin-converting enzyme. Biochem Pharma- col 61, 835–841. 18 Natesh R, Schwager SL, Sturrock ED & Acharya KR (2003) Crystal structure of the human angiotensin- converting enzyme-lisinopril complex. Nature 421 , 551– 554. 19 Cotton J, Hayashi MA, Cuniasse P, Vazeux G, Ianzer D, De Camargo AC & Dive V (2002) Selective inhibi- tion of the C-domain of angiotensin I converting enzyme by bradykinin potentiating peptides. Biochemis- try 41, 6065–6071. 20 Georgiadis D, Cuniasse P, Cotton J, Yiotakis A & Dive V (2004) Structural determinants of RXPA380, a potent and highly selective inhibitor of the angiotensin-convert- ing enzyme C-domain. Biochemistry 43, 8048–8054. 21 Michaud A, Williams TA, Chauvet MT & Corvol P (1997) Substrate dependence of angiotensin I-converting enzyme inhibition: captopril displays a partial selectivity for inhibition of N-acetyl-seryl-aspartyl-lysyl-proline hydrolysis compared with that of angiotensin I. Mol Pharmacol 51, 1070–1076. 22 Christlet THT & Veluraja K (2001) Database analysis of O-glycosylation sites in proteins. Biophys J 80, 952– 960. 23 Novikova EG, Eng FJ, Yan L, Qian YM & Fricker LD (1999) Characterization of the enzymatic properties of the first and second domains of metallocarboxypepti- dase D. J Biol Chem 274, 28887–28892. 24 Cal S, Quesada V, Llamazares M, Diaz-Perales A, Garabaya C & Lopez-Otin C (2005) Human polyserase- 2, a novel enzyme with three tandem serine protease domains in a single polypeptide chain. J Biol Chem 280, 1953–1961. 25 Binevski PV, Sizova EA, Pozdnev VF & Kost OA (2003) Evidence for the negative cooperativity of the two active sites within bovine somatic angiotensin-con- verting enzyme. FEBS Lett 550, 84–88. 26 Woodman ZL, Schwager SL, Redelinghuys P, Carmona AK, Ehlers MR & Sturrock ED (2005) The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity. Biochem J 389, 739–744. 27 Eisenthal R & Cornish-Bowden A (1974) The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J 139, 715–720. 28 Cornish-Bowden A & Eisenthal R (1974) Statistical con- siderations in the estimation of enzyme kinetic para- meters by the direct linear plot andother methods. Biochem J 139, 721–730. 29 Cornish-Bowden A & Eisenthal R (1978) Estimation of Michaelis constant and maximum velocity from the direct linear plot. Biochim Biophys Acta 523, 268–272. 30 Cornish-Bowden A, Porter WR & Trager WF (1978) Evaluation of distribution-free confidence limits for enzyme kinetic parameters. J Theor Biol 74, 163–175. 31 Kuzmic P (1996) Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem 237, 260–273. 32 Sali A & Blundell TL (1993) Comparative protein mod- elling by satisfaction of spatial restraints. J Mol Biol 234, 779–815. 33 Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S & Karplus M (1983) charm: a program for macromolecular energy, minimization, and calculat- ions. J Comput Chem 4, 187–217. 34 MacKerell AD Jr, Bashford D, Bellott M, Dunbrack RL Jr, Evanseck JD, Field MJ, Fischer S, Gao H, Ha S, Joseph-McCarthy D, et al. (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102, 3586–3616. N. D. Jullien et al. ACE substrate specificity FEBS Journal 273 (2006) 1772–1781 ª 2006 The Authors Journal compilation ª 2006 FEBS 1781 . Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme Nicolas. kcalÆ mol )1 ÆA ˚ )2 for the ions and their chelating residues, the atoms of the protein situated at a distance greater than 5 A ˚ of the inhibitor, and the backbone and Cb atoms