From the 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
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ABSTRACT |
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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 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.
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
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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 EXXD2
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.
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EXPERIMENTAL PROCEDURES |
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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 50% lower (Fig. 2B).
Glycosylation pattern was not affected by these mutations (Fig.
2A).
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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 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.
GT
,
Gbinding, and
Gcat, were calculated as described by
Wells (11).
GT
,
Gbinding, and
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·S
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·S
), respectively.
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RESULTS AND DISCUSSION |
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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
EXXD zinc-binding motif and is preceded by a
conserved Asp with 1 or 4 intervening residues. The distance between
the EX
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
EXXD zinc-binding motif (Fig.
3). The first of these, His1002, is 10 residues downstream of the
EX
XD motif and does not possess a nearby
upstream Asp. The second, His1089, is 97 residues
downstream of the EX
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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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 s1, 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·mol1 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
GT
of 3.8 kcal·mol
1 in transition state binding of
substrate (12), and in neprilysin the change in
GT
was 2.2 kcal·mol
1 (13). No change in phenotype was
observed with the His1002
Ala mutation (data not
shown), and thus further studies with this His mutant were not
performed.
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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·mol1 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-
-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-
-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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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
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
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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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·mol1 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
-CONHto -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
(GT
). The combined effect of
the two individual mutations on
GT
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
GT
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·mol1) 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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Summary and Conclusions--
We show that His1089 in
human ACE C-domain stabilizes the rate-limiting transition state by 3.7 kcal·mol1 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 -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 (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
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.
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FOOTNOTES |
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* 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
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).
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ABBREVIATIONS |
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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.
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REFERENCES |
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