Angiotensin I-converting Enzyme Transition State Stabilization by His1089

EVIDENCE FOR A CATALYTIC MECHANISM DISTINCT FROM OTHER GLUZINCIN METALLOPROTEINASES*

Marian FernandezDagger , Xifu LiuDagger , Merridee A. WoutersDagger , Sophie HeybergerDagger , and Ahsan HusainDagger §

From the Dagger  Enzyme Research Unit, Victor Chang Cardiac Research Institute, Sydney, New South Wales 2010 and the § School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, New South Wales 2033, Australia

Received for publication, October 3, 2000, and in revised form, October 31, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His1089 as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His1089 to Ala or Leu. At pH 7.5, with Ang I as substrate, kcat/Km values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (kcat) with minor effects on ground state substrate binding (Km). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His1089 right-arrow Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His1089 side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His1089 on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Angiotensin I (Ang I)1-converting enzyme (ACE, EC 3.4.15.1, peptidyl-dipeptidase A) is a chloride-activated peptidase with broad substrate specificity that releases a C-terminal dipeptide from substrates (1). The somatic form of human ACE has two homologous catalytic domains (1). These N- and C-domains most likely are the result of an ancient gene duplication event that occurred during vertebrate evolution (2). Invertebrate ACE has a single catalytic domain (3). The physiological substrates of ACE include Ang I, bradykinin, substance P, and AcSDKP. AcSDKP, the principal substrate of N-domain ACE, may play a role in hemopoietic cell differentiation, whereas both domains are thought to be important for regulating tissue and blood levels of the vasoactive hormones angiotensin II (Ang II) and bradykinin. Inhibition of these ACE activities has proved to be important in the treatment of hypertension and congestive heart failure (4).

ACE belongs to the gluzincin family (clan MA) of metalloproteases, of which thermolysin is the prototypical member (5). With thermolysin, catalysis occurs by a general base-type mechanism (6, 7). The proposed mechanism involves the displacement of a zinc-bound water molecule by the approaching substrate followed by the attack of this water molecule on the scissile carbonyl bond to form an oxyanion (Fig. 1). The attack of the water molecule is facilitated by the active site glutamic acid, Glu143. The resulting tetrahedral intermediate subsequently decomposes to yield the products. The negative charge on the scissile bond carbonyl oxygen that develops during transition state binding is stabilized by hydrogen bonding interactions with a protonated His, His231, and a tyrosine, Tyr157, in the active site. His231 is thought to be maintained in a protonated state through a hydrogen bonding interaction with Asp226. However, the functional consequence of the interaction between His231 and Asp226, inferred on the basis of crystallographic evidence, has not been confirmed by site-directed mutagenesis studies. The crucial stabilization of the oxyanion by His231 occurs after the formation of the Michaelis enzyme-substrate complex and greatly influences catalytic rate.



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Fig. 1.   Proposed mechanism of thermolysin-catalyzed hydrolysis. The reaction proceeds by the attack of the zinc-bound water molecule on the scissile bond. The resulting tetrahedral intermediate, with enzyme residues involved in its stabilization, is shown. This subsequently decomposes to yield the products. Adapted from Matthews (6). The nomenclature used for the individual amino acids (P1, P1', etc.) of a substrate and the subsites (S1, S1', etc.) of the enzyme is that of Schechter and Berger (34). Amino acid residues of substrates numbered P1, P2, etc. are toward the N-terminal direction, and P1', P2', etc. are toward the C-terminal direction from the scissile bond.

The catalytic mechanism in ACE is not known but has been inferred on the basis of that proposed for thermolysin. Some key active site residues in ACE have been determined because of similarity in local primary structure, such as those in the HEXXH and the EXtheta XD2 motifs. Thus it is known that His959, His963, and Glu987 are the zinc-ligating residues in human ACE C-domain, and Glu960 likely acts as a general base during catalysis (1, 8). Although it is realized that ACE is related by divergent evolution to thermolysin and neprilysin, key ACE residues involved in catalysis and substrate binding, other than the zinc-ligating residues and Glu960, have not been identified because the estimated overall primary sequence identity between ACE and other gluzincins is less than 15%. To construct structural models of ACE in the absence of a crystal structure, it is necessary to identify key, structurally conserved catalytic and substrate-binding residues so that the primary structures can be aligned with less ambiguity. Here we report the identification of the catalytic His, His1089, in human ACE C-domain equivalent to His231 in thermolysin. These studies indicate for the first time that the structure of ACE, C-terminal to the zinc-ligating residues, shows similarity to thermolysin and neprilysin. We show that the His1089 side chain allows ACE to function as an alkaline peptidyl-dipeptidase, and its interactions with inhibitors reveal major differences in binding modes when compared with other gluzincins.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Construction of Human ACE C-domain Gene-- A human ACE C-domain gene with 47 unique restriction sites was designed by strategies used previously (9) and was chemically synthesized and cloned into the shuttle expression vector pcDNA3 (Invitrogen). The synthetic gene encodes amino acids 1-29 (signal peptide) and 611-1201 of human somatic ACE with an 8-residue FLAG epitope recognized by a commercially available antibody (M2, Sigma) at the C terminus. This ACE synthetic gene does not include the transmembrane sequence found in somatic ACE. Mutations were constructed in the synthetic gene by cassette mutagenesis or site-directed mutagenesis, and all mutations were confirmed by DNA sequence analysis.

Transfection of COS-7 Cells-- COS-7 cells (ATCC) were cultured under an atmosphere of 5% CO2 at 37 °C and transfected with plasmid DNA using the Gene Pulser system (Bio-Rad). 12 h after transfection, cells were washed and further cultured with serum-free Dulbecco's modified Eagle's media (Life Technologies, Inc.) for 48-72 h. C-domain ACE and its mutants released into conditioned media were collected and used as the starting point of the purification. The following ACE C-domain mutations were made: H1002A, H1089A, H1089L, D1083A, D1083N, and D1083A/H1089A. Fig. 2 shows the effect of these mutations on glycosylation patterns (Western blot) and protein expression levels. A polyclonal antiserum generated against pure human kidney ACE (gift from Dr. R. Ramchandran, Cleveland Clinic Foundation) was used in Western blots. Expression levels of H1089A and H1089L proteins by COS-7 cells was similar to that of wild-type ACE C-domain and that of D1083A, D1083N, and D1083A/H1089A was approx 50% lower (Fig. 2B). Glycosylation pattern was not affected by these mutations (Fig. 2A).



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Fig. 2.   Immunoblot analysis of protein expressed in COS-7 cell extracellular extracts. A, Western blot of COS-7 cell extracellular extracts. Extracellular extracts were as follows: lane 1, wild-type ACE C-domain; lane 2, H1089A-ACE C-domain; lane 3, H1089L-ACE C-domain; lane 4, D1083A-ACE C-domain; lane 5, D1083N-ACE C-domain; lane 6, D1083A/H1089A-ACE C-domain. B, slot-blot quantitation of COS-7 cell extracellular extracts. Values are ACE C-domain-like immunoreactivity/total protein in the extracellular extract.

Purification of Recombinant Human C-domain ACE and Its Mutants-- An anti-FLAG M2 affinity gel was used to purify human C-domain ACE according to the procedure described by the manufacturer (Sigma). This ACE protein preparation was >95% pure as determined by Coomassie Blue staining, and the protein amount was quantified by amino acid analysis. This preparation of human ACE C-domain was used as a standard. For kinetic studies, human C-domain ACE and its mutants were partially purified by ion-exchange HPLC using a Bio Scale Q2 column (Bio-Rad). Cell culture media containing ACE C-domain or its mutant was dialyzed against 20 mM Tris-HCl buffer, pH 7.5, containing 20 mM NaCl. The dialyzed media (5-10 ml) was applied to the ion-exchange HPLC column which was developed using a 45-min linear NaCl gradient (20-500 mM) at a flow rate of 1 ml/min. Two to three 1-ml fractions with the highest activity were pooled. The purity of recombinant ACE in the pooled peak fractions was approx 50%, with bovine serum albumin as the only detectable contaminant. ACE was not further purified to remove the contaminating bovine serum albumin since the stability of pure (>95%) ACE C-domain at 4 °C was markedly lower than that of partially purified ACE containing bovine serum albumin.

Peptides-- Peptides used in this study were synthesized by The Protein Core Facility, The Cleveland Clinic Foundation or Auspep (Parkville, Australia). Peptides were purified (purity >99%) on a C18 reverse phase HPLC column and characterized by amino acid analysis and by analytical C18 reverse phase HPLC. Peptide concentrations were determined by amino acid analysis.

Enzymes and Enzyme Kinetics-- To determine Km and Vmax values for human ACE C-domain reactions, initial velocities (v) were determined as described by us previously (10). Twelve concentrations of Ang I ranging between 5 and 250 µM were incubated with wild-type or mutants forms of ACE at 37 °C in 50 mM HEPES buffer, pH 7.5, containing 50 mM NaCl and 10 µM ZnSO4 (final volume 50 µl) for 30-60 min. Enzyme concentration was adjusted to between 0.1 and 16 nM to ensure that <15% of the substrate was consumed at the lowest substrate concentration. Under these conditions, product formation was linear with respect to time over the duration of the incubation. For pH dependence studies, AcAla-Phe-Ala-Ala-COOH was used as substrate at a concentration (40 µM) 10-fold lower than its Km value, and thus variations in activity with respect to pH reflect changes in kcat/Km values. Buffers used in pH studies3 are as follows: 50 mM sodium acetate containing 10 mM ZnSO4, 50 mM MES containing 0.1 mM ZnSO4, 50 mM HEPES containing 10 µM ZnSO4, and 50 mM CHES containing 10 µM ZnSO4 for the pH ranges 3.4-5.6, 5.5-6.9, 6.7-8.8, and 8.4-10.5, respectively. Reactions were terminated by the addition of 70 µl of ice-cold 0.1% trifluoroacetic acid or 2% H2PO4. The resulting solution (100 µl) was applied to a C18 reverse phase HPLC column (XTerra RP18 3.5 µm, 4.6 × 50 mm column, Waters, Milford, MA). The column was developed with linear acetonitrile gradients containing either 25 mM triethylammonium phosphate buffer, pH 3.0 or 7.0, or 0.1% trifluoroacetic acid at a flow rate of 2 ml/min. The column effluent was monitored at 214 nm. The elution positions of Ang I, Ang II, AcAla-Phe-Ala-Ala-COOH, and AcAla-Phe-COOH were determined using pure synthetic standards. The peak area corresponding to Ang II or AcAla-Phe-COOH was integrated to calculate product formation. Products were separated by reverse phase HPLC and identified by amino acid analysis. Km and Vmax values were calculated by nonlinear regression using the equation v = Vmax × [S]/(Km + [S]). Correlation coefficients were routinely >0.99 but never <0.97.

Assays with inhibitors were performed in 50 mM HEPES buffer, pH 7.5, containing 50 mM NaCl and 10 µM ZnSO4 at 37 °C with 40 µM Ang I as substrate in a total volume of 50 µl; incubation period was 30 min. Prior to the addition of substrate (5 µl), enzyme was preincubated with inhibitors for 1 h. Ki values were determined from plots of enzyme activity versus inhibitor concentration where inhibitor concentration was corrected by using the Km and concentration of the substrate used for the competition (Ki = IC50/(1 + ([substrate]/Km))).

Pure human wild-type ACE C-domain of known concentration was used in a Western blot protocol to determine enzyme concentration of partially purified ACE and mutant ACE enzyme preparations. The overall rate constant kcat was calculated by the formula kcat Vmax/[E0], where [E0] is the total enzyme concentration. Delta Delta GTDagger , Delta Delta Gbinding, and Delta Delta Gcat, were calculated as described by Wells (11). Delta Delta GTDagger , Delta Delta Gbinding, and Delta Delta Gcat represent the difference between two enzymes in the free energy required for transition state stabilization (i.e. difference in free energy required to reach E·SDagger from E + S), to form the enzyme-substrate complex (E·S) from E + S, and to convert the E·S complex to the transition state complex (E·SDagger ), respectively.


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Prediction of His1089 as a Transition State Stabilizing Residue in Human ACE C-domain-- In thermolysin, thermolysin-like enzymes, and neprilysin, in which the identity of the catalytic His has been confirmed by site-directed mutagenesis (12, 13) and x-ray crystallography (14, 15), this residue is found C-terminal to the EXtheta XD zinc-binding motif and is preceded by a conserved Asp with 1 or 4 intervening residues. The distance between the EXtheta XD motif and the transition state His is 49-60 residues in vertebrate neprilysin-like and bacterial thermolysin-like sequences.

Human ACE C-domain possesses two conserved histidines C-terminal to the EXtheta XD zinc-binding motif (Fig. 3). The first of these, His1002, is 10 residues downstream of the EXtheta XD motif and does not possess a nearby upstream Asp. The second, His1089, is 97 residues downstream of the EXtheta XD motif and is preceded by an Asp with 5 intervening residues. A conserved Pro two residues downstream of the catalytic His in neprilysin-like sequences is also found two residues downstream of His1089 in vertebrate ACEs. On the basis of these features, we predicted and tested if His1089 is the transition state stabilizing His.



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Fig. 3.   Alignment of several gluzincins. Sequence alignment (partial sequences) of representative members of the M13 (neprilysin, NEP; endothelin-converting enzyme, ECE), ACE, and M4 (pseudolysin, ELAS; neutral proteinase, NPRE; thermolysin, THER) families of gluzincins. Zinc-binding motifs (HEXXH, EXtheta XD) and transition state His motif (D(X)nH) are boxed. Numbering at the top is that of the human ACE C-domain. Sequence numbers for other sequences are shown at the right. Zinc-binding residues are indicated by  and catalytic residues are indicated by Delta . The prefixes h, r, dm, hi, pa, bc, and bt refer to human, rat, Drosophila melanogaster, Hematobia irritans, Pseudomonas aeruginosa, Bacillus cereus, and Bacillus thermoproteolyticus, respectively.

Design and Properties of Human ACE C-domain-- Human ACE has two fully functional catalytic domains, N and C. To assign function to individual residues in human ACE through kinetic analyses, it is necessary to express each domain separately or to inactivate the domain not under study. Both approaches have been used previously (16). We constructed a synthetic human ACE C-domain gene consisting of exons 14-25 (coding for residues 611-1201) of the human ACE gene. This region is homologous to invertebrate ACE and to exons 1 (excluding the signal peptide + 6 residues) to 12 of the human ACE gene, that is the ACE N-domain. The human ACE C-domain construct contains the catalytic residues and sequence up to the region that is reported to be cleaved during ACE shedding from the plasma membrane but does not contain the unique C-terminal transmembrane spanning and cytosolic domains encoded by exon 26. Km, kcat, and kcat/Km values for Ang I conversion to Ang II by human ACE C-domain were 21 ± 2.5 µM, 18.8 ± 1.8 s---1, and 0.9 µM·s-1, respectively (n = 3). These values are similar to those obtained for Ang I to Ang II conversion by full-length human ACE where the N-domain is inactivated through mutation of the zinc-ligating residues (17).

Role of His1089 in Ground State and Transition State Substrate Binding-- Substrate binding to ground and transition states of wild-type ACE C-domain and its mutants was studied using Ang I. The results are summarized in Table I. The His1089 to Ala or Leu mutations in human ACE C-domain produced a small increase in Km and a large decrease in kcat values. A loss in transition state binding energy of 3.74 kcal·mol-1 is associated with the His1089 to Ala mutation and 5.13 kcal·mol-1 with the Leu mutation. This loss of transition state binding energy is consistent with the loss of a strong hydrogen bond in the tetrahedral intermediate as would be expected between a strong hydrogen bond donor and acceptor. Equivalent mutations in a thermolysin-like enzyme produced a change in Delta GTDagger of 3.8 kcal·mol-1 in transition state binding of substrate (12), and in neprilysin the change in Delta GTDagger was 2.2 kcal·mol-1 (13). No change in phenotype was observed with the His1002 right-arrow Ala mutation (data not shown), and thus further studies with this His mutant were not performed.


                              
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Table I
Hydrolysis of Ang I by wild-type and His1089 and Asp1083 mutants of human ACE C-domain
Kinetic constants were determined in 50 mM HEPES buffer, pH 7.5, containing 50 mM NaCl and 10 µM ZnSO4 at 37 °C. Km and kcat values were determined by nonlinear regression; values are means ± S.E. of three independent determinations for each peptide. Values in parentheses represent Delta Delta G values. Delta Delta GTDagger , Delta Delta Gbinding, and Delta Delta Gcat represent the difference between two enzymes in the free energy required for transition-state stabilization (i.e. difference in free energy required to reach E · SDagger from E + S), to form the enzyme---substrate complex (E · S) from E + S, and to convert the E · S complex to the transition-state complex (E · SDagger ), respectively.

A key feature of the catalytic His in gluzincins is that it greatly impacts transition state binding with minimal effects on ground state binding. If Km is equal to the true dissociation constant (Ks) for the enzyme and substrate, the 4.5-fold increase in Km associated with the His1089 to Ala mutation would correspond to a 0.93 kcal·mol-1 loss in binding energy of the substrate to the ground state structure. The effect of the His1089 to Ala mutation on ground state structure of the enzyme was also examined using [Phe8-psi -His9]Ang I, an Ang I analog where the scissile amide group (-CONH-) in Ang I is replaced by a aminomethylene (-CH2NH-) isostere, so that essential contacts required for transition state binding are absent (Fig. 4, A and B). Loss of [Phe8-psi -His9]Ang I binding energy associated with the His1089 to Ala substitution was 0.05 kcal·mol-1 (Fig. 5A and Table II). These findings indicate that ground state structure is not altered in the His1089 to Ala mutant.



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Fig. 4.   Presumed binding modes of Ang I and inhibitors used in this study to the active site of ACE C-domain. Expected interactions with the zinc atom, based on studies with thermolysin (6, 19, 20, 22, 23), and those based on the findings of the present study are shown by dotted lines. A, Ang I; B, [Phe8-psi -His9]Ang I; C, captopril; D, lisinopril. The dotted green lines represent bonds based on studies with thermolysin and on our kinetic studies with wild-type and H1089A-ACE C-domain. The dotted red line in C represents a hydrogen bond based on our kinetic studies with wild-type and H1089A-ACE C-domain and is not observed in thermolysin.



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Fig. 5.   Inhibition of wild-type (, solid line) and H1089A-ACE C-domain (open circle , dotted line) by ACE inhibitors. A, [Phe8-psi -His9]Ang I; B, captopril; C, lisinopril. Assays with inhibitors were performed in 50 mM HEPES buffer, pH 7.5, containing 50 mM NaCl and 10 µM ZnSO4 at 37 °C with 30 µM Ang I as substrate in a total volume of 50 µl; incubation period was 30 min. Prior the addition of substrate (5 µl) enzyme was preincubated with inhibitors for 1 h. Values are means ± S.E. of three independent determinations for each inhibitor. Km values for wild type and H1089A used in abscissa calculation were from Table I.


                              
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Table II
Delta Delta G values of inhibitor binding between wild-type ACE C-domain and H1089A and D1083A
Values represent the difference in free energy (Delta Delta G) between wild-type and mutant enzyme and were calculated using the equation Delta Delta G = -RT · ln(Ki(wild-type)/Ki((mutant)). Ki values are from Figs. 5 and 7.

His1089 and Glu960 in ACE C-domain play an important role in catalysis. The pKa values of the His imidazole group and the Glu side chain carboxylate group are expected to be in the range 5 to 8 and 2 to 5.5, respectively (18). To study the role of these residues in determining pH dependence of activity, we used the artificial ACE substrate AcAla-Phe-Ala-Ala-COOH. This substrate is preferable to Ang I in that it has no ionizable groups in the pH range 6-9. The pH optimum of ACE C-domain-dependent cleavage of AcAla-Phe-Ala-Ala-COOH is approx 8 (Fig. 6A). It is therefore expected that pH dependence of ACE C-domain activity at around neutral pH will be greatly influenced by His1089. Indeed, pH dependence of activity in the pH range 6.5-10.5 was not evident in H1089A, and the mutant was approx 100-fold less active than wild type (Fig. 6B). Thus, His1089 is a major determinant in phenotype in human ACE C-domain that is responsible for the alkaline pH optimum of this enzyme. In H1089A, a sharp pH optimum was observed at 5.5 (Fig. 6B). The maximal activity at pH 5.5 with H1089A was similar to that observed with wild-type enzyme at this pH. The H1089A mutation likely unmasks the pKa of the remaining ionizable group, and the sharp increase in activity between pH 5 and 5.5 is likely due to the deprotonation of the carboxylate side chain of Glu960 that facilitates the attack of the zinc-bound water on the peptide carbonyl bond (Fig. 4A). The rapid quenching of activity between pH 5.5 and 6 was unexpected and suggests that deprotonation of a carboxylate other than that of Glu960 hinders catalysis. This observation differs markedly from that seen with a thermolysin-like enzyme where the pH optimum shifts from 6 to 8 when a similar mutation is introduced in place of the catalytic His (12).



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Fig. 6.   pH dependence of wild-type (A), H1089A- (B), and D1083A-ACE C-domain (C) activity. Assays were performed with the substrate AcAla-Phe-Ala-Ala-COOH (40 µM) as described under "Experimental Procedures." Substrate concentration used was 10-fold lower than Km, and therefore changes in activity reflect changes in kcat/Km. Buffers used were sodium acetate (), MES (), HEPES (open circle ), and CHES (black-square) at a concentration of 50 mM containing 10 and 0.1 mM, and 10 and 10 µM ZnSO4, respectively. The gray box in A-C represents the pH optimum of wild-type ACE C-domain.

Role of His1089 in Captopril and Lisinopril Binding-- Several classes of metalloprotease inhibitors have been described, and their mode of binding with thermolysin and thermolysin-like enzymes has been studied by crystallography. The different classes of inhibitors studied include the mercaptans (19, 20), hydroxymates (21), carboxylates (22, 23), and phosphoramidates (24). All these inhibitors are coordinated to the catalytic zinc, but interactions with the transition state stabilizing residues His231 and Tyr157 differ. Carboxylates and phosphoramidates are hydrogen-bonded to His231 and Tyr157. Hydroxymates form a hydrogen bond with His231 but do not interact with Tyr157, whereas mercaptans do not display hydrogen bonding interactions with either of these residues. Mutation studies with thermolysin have confirmed the presence or absence of these interactions with His231. To examine the similarities and differences between ACE and other gluzincins, we studied the His1089 interaction with two distinct inhibitors of human ACE as follows: lisinopril, a carboxylate inhibitor, and captopril, a mercaptan inhibitor.

The predicted mode of binding of lisinopril to the ACE C-domain is shown in Fig. 4D and is based on crystallographic studies with thermolysin. The His1089 to Ala mutation produced a 7.2 kcal·mol-1 loss in the binding energy of lisinopril (Fig. 5C and Table II) suggesting that the bidentate mode of binding of the carboxylate group resembles the presumed geometry of the tetrahedral transition state, with a hydrogen bond being formed between one oxygen of the carbonyl group and His1089. Unexpectedly, the His1089 to Ala mutation produced a 5.3 kcal·mol-1 loss in the binding energy of captopril (Fig. 4B and Table II) suggesting a direct hydrogen bonding interaction between the imidazole N-H and the captopril sulfur, presumably in the anionic form (23). Fig. 4C illustrates the presumed binding scheme. N-H····S hydrogen bonds are described in protein data bases (25, 26). Because S is a weak hydrogen bond acceptor, N-H···· hydrogen bonds are generally weak (<-1.2 kcal·mol) (27); however, strong hydrogen bonds with S as an acceptor have also been described (28) and could operate in ACE C-domain-captopril complexes.

Previous studies have shown differences between inhibitor binding to thermolysin and neprilysin, as compared with ACE. For example, retro-inverso modification of the amide bond (from -CONH---to -NHCO-) has little effect on inhibitory potencies of mercaptan inhibitors of thermolysin and neprilysin, but this change produces a marked decrease (>1,000-fold) in ACE inhibitor potency (12, 29). These findings illustrate differences in the substrate binding pocket of these gluzincins distal to the site that interacts with the scissile carbonyl bond. Our findings with captopril show for the first time that atoms arranged around the tetrahedral carbon designed to mimic the transition state of the substrate interact in a markedly different manner in ACE compared with thermolysin-like enzyme and neprilysin.

Does Asp1083 Interact with His1089?-- In several gluzincin crystal structures, including those of thermolysin and neprilysin, the transition state His is located in close proximity to an Asp side chain, such that a hydrogen bonding network between these residues has been proposed (15, 30). It is believed that this interaction appropriately positions the imidazole side chain and maintains it in a protonated state. In gluzincins with known structures, this Asp is located 2-5 residues N-terminal to the transition state His in the primary structure, but the carboxyl head group superimposes in the tertiary structure. In ACE C-domain, a conserved Asp, Asp1083, is located 6 residues N-terminal to His1089. To study if Asp1083 interacts functionally with His1089, the effects of single His and Asp mutations to Ala were compared with the double mutant D1083A/H1089A. The results are summarized in Table I. The main effect of the Asp1083 to Ala mutation was on kcat resulting in a 1.8 kcal·mol-1 loss in transition state binding energy (Delta Delta GTDagger ). The combined effect of the two individual mutations on Delta Delta GTDagger was additive to within 7% of that observed in the double mutant indicating that these residues stabilize the transition state by independent mechanisms. A potential hydrogen bonding interaction between Asp1083 and His1089 should also influence the pKa of the catalytic imidazole group. If there is such an interaction in ACE C-domain, analogous to that in thermolysin and neprilysin, the loss of this Asp interaction should acid-shift the pH optimum of ACE C-domain. The pH optimum of D1083A, however, is not lower than that of the wild-type enzyme (Fig. 6C). This indicates again that Asp1083 does not influence the orientation or protonation state of the His1083 side chain, as is evident from Delta Delta GTDagger additivity analyses.

The chief effect of the Asp1083 to Ala or Asn mutation is on transition state binding, since these mutants display a 1.8 or 3.1 kcal·mol-1 loss in Ang I binding energy, respectively (Table I). However, these mutations minimally affect either captopril binding or lisinopril binding (0.91 kcal·mol-1) which mimics the tetrahedral intermediate insofar as the scissile bond is concerned (Fig. 7 and Table II). These findings indicate that in the transition state, Asp1083 is directly or indirectly involved in making contacts between Ang I and ACE C-domain but is not involved in those interactions that are directed toward the scissile bond. In producing this effect, Asp1083 appears to make important ionic interactions since the Asp1083 to Asn mutation, which retains hydrogen bonding potential but is unable to make ion pair interactions, was unable to mimic the requirement of Asp1083 in transition state substrate binding (Table I). In these respects, ACE differs significantly from thermolysin and neprilysin where Asp226-His231 and Asp709-His711 interactions are observed, respectively, in the crystal structure (15, 30). Moreover, ACE differs from other gluzincins such as Vibrio anguillarum vibriolysin, where the Asp equivalent to Asp226 of thermolysin is naturally replaced by Asn.



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Fig. 7.   Inhibition of wild-type (solid line), D1083A- (, dotted line), and D1083N-ACE C-domain (open circle , dotted line) by ACE inhibitors. A, captopril; B, lisinopril. For conditions see Fig. 5 legend. Values are means ± S.E. of three independent determinations for each inhibitor. Km values for wild-type, D1083A, and D1083N used in abscissa calculation were from Table I.

Summary and Conclusions-- We show that His1089 in human ACE C-domain stabilizes the rate-limiting transition state by 3.7 kcal·mol-1 with minor effect on the ground state structure. The catalytic His is known to stabilize the transition state by making a hydrogen bond with the oxyanion in the tetrahedral intermediate. In human ACE C-domain, this interaction, schematically illustrated in Fig. 4A, has a binding energy equivalent to that of a strong hydrogen bond. A similar loss of binding energy of lisinopril, an inhibitor of ACE that closely mimics the transition state configuration of the scissile bond, is observed with the His1089 to Ala mutation. The presence of an Asp1083-(Xaa)5-His1089 motif in human ACE C-domain equivalent to that in other gluzincins and the selective effect of the His1089Ala mutation on transition state binding imply that His1089 in human ACE C-domain is the catalytic His equivalent to His231 in thermolysin and His711 in neprilysin. Because of the high homology between the N- and C-domains of ACE, it is likely that the equivalent residue in human ACE N-domain, His491, is also a catalytic residue.

The overall structure of thermolysin consists of two roughly spherical lobes with a deep cleft between the two that is used to coordinate the catalytic zinc and to bind the substrate (31). Helical structure predominates in the C-terminal lobe, whereas beta -structure predominates in the N-terminal lobe. Although neprilysin is a much larger protein, the crystal structure shows it to be structurally similar to thermolysin in the C-terminal half of the N-terminal lobe and most of the C-terminal lobe (15). These regions surround the active site and include the zinc-ligating residues and transition state His. Based on the presence of near-identical primary structural motifs for the zinc-ligating residues in ACE N- and C-domains and thermolysin, it is expected that tertiary structure of ACE around the zinc-binding sites will be conserved. The prediction that His1089 is a catalytic residue in ACE was initially based on multiple sequence alignment. This alignment is likely to be largely correct on the basis of our experimental findings and suggests for the first time a more extended conservation between the C-terminal lobe tertiary structure of thermolysin, neprilysin, and ACE.

The pH optimum of human ACE (approx 8.0) is higher than that reported for other gluzincins (5.5 to 7.5). pH dependence of human ACE C-domain activity in the pH range 6.5 to 9 is entirely due to His1089, such that H1089A is an acid peptidyl-dipeptidase with a pH optimum approx 5.5. The marked effect of the catalytic His on pH dependence is an unusual design feature of this gluzincin. The mode of binding of inhibitors to thermolysin has been well documented (19-24) and has served as a model of how the different classes of inhibitors bind to gluzincins. Here we provide evidence for a marked difference in the binding mode of a mercaptan to ACE. These variations in the binding pocket suggest that it is possible to design new types of ACE inhibitors, not previously anticipated from studies with other gluzincins. These differences between ACE and thermolysin in the role of the catalytic His in influencing the pH optimum and in the mode of binding of transition state inhibitors question the general use of thermolysin-based mechanistic models for predicting structure-based function in gluzincins.


    FOOTNOTES

* This work was supported by NH&MRC Grants 109001 and 990990.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Enzyme Research Unit, Victor Chang Cardiac Research Institute, 384 Victoria St., Sydney, New South Wales 2010, Australia. Tel.: 612-9295-8507; Fax: 612-9295-8509; E-mail: a.husain@victorchang.unsw.edu.

Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M009009200

2 theta is usually aliphatic but sometimes is aromatic.

3 ZnSO4 concentrations were increased progressively as the buffer pH was reduced, because zinc begins to dissociate from the enzyme below pH 7 (32). The concentrations of zinc used were those used by Shapiro et al. (33).


    ABBREVIATIONS

The abbreviations used are: Ang I, angiotensin I; Ang II, angiotensin II; ACE, angiotensin I-converting enzyme; HPLC, high performance liquid chromatography; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; MES, 2-[N-morpholino]ethanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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