Báo cáo khoa học: Structural diversity of angiotensin-converting enzyme Insights from structure–activity comparisons of two Drosophila enzymes docx

Other differences in the S2¢ site of ANCE and ACER also explain the selec-tivity of RXPA380, a selective inhibitor of human C-domain ACE, which also preferentially inhibits ACER.. Anothe

Insights from structure–activity comparisons of two Drosophila enzymes

Richard J Bingham1, Vincent Dive2, Simon E V Phillips1, Alan D Shirras3and R Elwyn Isaac1

1 Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, UK

2 Departement d’Etudes et d’Ingenierie des Proteines, Commissariat a l’Energie Atomique, CE-Saclay, Gif-Sur-Yvette, France

3 Department of Biological Sciences, University of Lancaster, UK

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is

a zinc peptidyl-dipeptidase, which is best known for

catalysing the last step in the synthesis of the

vasocon-strictor angiotensin II (AII) from angiotensin I (AI)

and for the metabolic inactivation of the vasodilator

bradykinin (BK) [1] The somatic form of the enzyme

is a glycosylated type I membrane protein comprising two homologous domains, generally known as the N-domain and C-domain, arranged in tandem and joined by a short connecting peptide sequence [2]

Keywords

ACE inhibitors; angiotensin-converting

enzyme (ACE); Drosophila melanogaster;

peptide metabolism; peptidyl-dipeptidase

Correspondence

R E Isaac, Faculty of Biological Sciences,

Miall Building, University of Leeds,

Leeds LS2 9JT, UK

Fax: +44 113 34 32835

Tel: +44 113 34 32903

E-mail: r.e.isaac@leeds.ac.uk

(Received 21 September 2005, revised

15 November 2005, accepted 21 November

2005)

doi:10.1111/j.1742-4658.2005.05069.x

The crystal structure of a Drosophila angiotensin-converting enzyme (ANCE) has recently been solved, revealing features important for the binding of ACE inhibitors and allowing molecular comparisons with the structure of human testicular angiotensin-converting enzyme (tACE) ACER is a second Drosophila ACE that displays both common and dis-tinctive properties Here we report further functional differences between ANCE and ACER and have constructed a homology model of ACER to help explain these The model predicts a lack of the Cl–-binding sites, and therefore the strong activation of ACER activity towards enkephalinamide peptides by NaCl suggests alternative sites for Cl– binding There is a marked difference in the electrostatic charge of the substrate channel between ANCE and ACER, which may explain why the electropositive peptide, MKRSRGPSPRR, is cleaved efficiently by ANCE with a low Km, but does not bind to ACER Bradykinin (BK) peptides are excellent ANCE substrates Models of BK docked in the substrate channel suggest that the peptide adopts an N-terminal b-turn, permitting a tight fit of the peptide in the substrate channel This, together with ionic interactions between the guanidino group of Arg9 of BK and the side chains of Asp360 and Glu150

in the S2¢ pocket, are possible reasons for the high-affinity binding of BK The replacement of Asp360 with a histidine in ACER would explain the higher Km recorded for the hydrolysis of BK peptides by this enzyme Other differences in the S2¢ site of ANCE and ACER also explain the selec-tivity of RXPA380, a selective inhibitor of human C-domain ACE, which also preferentially inhibits ACER These structural and enzymatic studies provide insight into the molecular basis for the distinctive enzymatic fea-tures of ANCE and ACER

Abbreviations

ACE, angiotensin-converting enzyme; ANCE, Drosophila melanogaster angiotensin-converting enzyme; ACER, Drosophila melanogaster angiotensin-converting enzyme-related; BK, bradykinin; AI, angiotensin I; AII, angiotensin II; Abz, o-aminobenzoic acid; Hip-His-Leu,

hippuryl-L -histidyl- L -leucine.

Each domain is catalytically active, and both are

cap-able of cleaving AI and BK The ACE gene also gives

rise to a second mammalian ACE, known as either

tes-tis (tACE) or germinal ACE, through the use of an

in-tragenic promoter that drives expression in developing

spermatocytes It is a single-domain enzyme that is

identical with the C-domain of somatic ACE, apart

from a peptide insert encoded by the testis-specific

exon 13 of the ACE gene [2] ACE knockout mice

dis-play renal abnormalities, low blood pressure, anaemia

and male infertility, confirming the important role of

this enzyme in development, blood homoeostasis and

reproduction [2]

Although N-domain and C-domain are highly similar

in protein sequence and share many enzymatic

proper-ties, they can be differentiated by substrate and

inhib-itor preferences and by the extent to which they are

activated by Cl– [3–5] The haemoregulatory peptide,

N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), another in vivo

substrate for mammalian ACE, is hydrolysed more

effi-ciently by the N-domain, as is the internally quenched

fluorogenic substrate Abz-SDK(Dnp)P [6,7] Cl– can

stimulate the activity of both ACE domains, but the

C-domain active site is more sensitive to changes in

Cl–concentration [3] The level of activation, as well as

the concentration of Cl– required for maximal

stimula-tion, is dependent on pH and the peptide substrate The

two domains of mammalian ACE can also be

distin-guished by the N-domain-selective inhibitor RXP407

[8], the C-domain-selective inhibitor RXPA380 [9], and

several BK-potentiating peptides [10]

A homologue of ACE, known as ACE2, has been

characterized as a single-domain type I glycoprotein

[11,12] It is important for normal contractility of heart

muscle [13] The important enzymatic feature of ACE2

is that, unlike ACE, it is a carboxypeptidase, removing

a single residue from the C-terminus of peptides that

have either a Pro or Leu in the P1 position, e.g

angio-tensin II, apelin 13 and dynorphin A 1–13 [14] The

activity of ACE2 is greatly enhanced in the presence

of NaCl [15,16] Therefore Cl– activation is a common

feature of the mammalian members of the ACE family

of peptidases

In vertebrates, the number of ACE genes appears to

be limited to ACE and ACE2, but in some insects

there has been a much greater expansion of this gene

family For example, in the mosquito, Anopheles

gambiae, and in Drosophila melanogaster there are nine

and six ACE genes, respectively [17,18] Of the six

Drosophila genes, only ANCE and ACER have been

confirmed to produce functional metallopeptidases

[19,20] They are both single-domain proteins with

40% amino-acid sequence identity and 60%

similar-ity to each of the two domains of mammalian ACE ANCE and ACER have distinct tissue expression pat-terns, indicating different physiological roles [21,22] ANCE appears to have a role in embryogenesis, meta-morphosis and reproduction [20,23,24] A function for ACER has not been established, but the protein is associated with the developing heart in embryos and in the brain and reproductive tissues of adults (A Carhan, R.E Isaac and A.D Shirras, unpublished results) The two enzymes share some enzymatic prop-erties, such as peptidyl-dipeptidase activity towards hippuryl-l-histidyl-l-leucine (Hip-His-Leu), and BK, and inhibition by inhibitors of mammalian ACE [19,20,25] However, compared with ANCE, ACER displays more restricted substrate specificity Although both ANCE and ACER hydrolyse Hip-His-Leu, only the ANCE activity is enhanced in the presence of NaCl [20,25] Another interesting difference between ACER and ANCE is that the ACER active site, but not that of ANCE, can accommodate an N-domain-specific inhibitor (RXP407), indicating common active-site features for ACER and the N-domain of human ACE [17]

Recent descriptions of the high-resolution molecular structure of ACE–inhibitor complexes for both human tACE [26,27] and Drosophila ANCE [28] have revealed the molecular details of the active site and how ACE inhibitors bind with high affinity These studies con-firm many of the predictions regarding the identity of the active-site residues and, in the case of tACE, iden-tify other side chains involved in the binding of Cl– at two sites (Cl1 and Cl2) positioned outside of the active site The crystal structure of human ACE2, with and without bound inhibitor, has also recently been repor-ted [29] and has provided a structural explanation for why ACE2 is a carboxypeptidase and not a peptidyl-dipeptidase The structure of the native ACE2 identi-fied a single Cl–-binding site that corresponded to the Cl1 site of tACE No bound Cl– was recognized in the crystal structure of Drosophila ANCE, and it has been proposed that the equivalent Cl–-binding sites in ANCE are substantially different and, in the case of Cl2, may be absent [26], which may explain the weaker effect of Cl– on enzyme activity reported for this enzyme In ACE2, the Cl2 site also does not exist, which leaves only Cl1 as a recognized Cl–-binding site [29] Interestingly, an alternative, but undefined, bind-ing site for Cl–has been suggested, which may be influ-ential in the conformational movement that occurs on formation of the ACE2 ES complex [26,29]

Comparative molecular and biochemical studies of members of the ACE family are likely to provide new insights into the evolution of the ACE active site, the

structural basis for differences in substrate specificity

and the mechanisms by which Cl– can have profound

effects on enzyme activity In this respect, Drosophila

ANCE and ACER appear to be good examples of two

family members that have diverged in structure and

substrate specificity and are therefore likely to provide

valuable information We now report on additional

biochemical differences between ANCE and ACER

regarding substrate specificity, the effect of Cl– on

enzyme activity, and inhibition by new

domain-select-ive inhibitors of human ACE A model of the structure

of ACER has been generated, which provides

explana-tions for some of these biochemical differences

Results

Hydrolysis of AI

The effect of NaCl on the conversion of AI into AII by

ANCE was determined at two pH values At pH 7,

increasing the concentration of NaCl resulted in a faster

rate of conversion, which reached a plateau at 150–

200 mm NaCl (Fig 1A) At pH 8, maximal activity was

achieved in the absence of NaCl, which had a weak

inhibitory effect on the hydrolysis of AI as the salt

con-centration increased from 0 to 200 mm NaCl (Fig 1A)

To further examine the effect of NaCl and pH on the

mechanism of ANCE activation, the kinetic constants

of AI hydrolysis were determined in the presence and

absence of 100 mm NaCl at pH 7 and 8 (Table 1) The

activation by NaCl at pH 7 was the result of a 330%

increase in kcat⁄ Km, which was solely attributable to a

lowering of the Km A similar rise in the kcat⁄ Km was

observed when the pH was increased from 7 to 8 in the

absence of NaCl, but in this case the greater catalytic

efficiency was achieved by a combined increased kcat

and a lower Km Although AI is an extremely poor

sub-strate for ACER, it was possible to determine kinetic

constants for this reaction (Km 1.58 ± 0.28 mm; kcat

0.01 ± 0.001 s)1), which showed that this marked

dif-ference between ACER and ANCE was due to the very

low kcatfor AI hydrolysis by ACER This weak

pept-idyl-dipeptidase activity, unlike that of ANCE and

mammalian ACE, was not stimulated by NaCl

(Table 2)

Hydrolysis of enkephalin peptides

[Leu5]Enkephalin, [Met5]enkephalin and their

respect-ive C-terminal amidated forms are hydrolysed at the

Gly-Phe bond by both ANCE and ACER at neutral

pH [20] The endopeptidase activity of ACER, but

not ANCE, towards [Leu5]enkephalinamide and

[Met5]enkephalinamide was stimulated in the presence

of Cl– ions (Table 2) The enhancement of the hydro-lysis of the amidated peptides by 500 mm NaCl was 12-fold and 15-fold, respectively, whereas the cleavage

of both [Leu5]enkephalin and [Met5]enkephalin was inhibited by
50% (Table 2) The NaCl-induced activ-ity of ACER was measured at different [Leu5]enkeph-alinamide and [Met5]enkeph[Leu5]enkeph-alinamide concentrations, which generated anomalous kinetics, including sub-strate inhibition at peptide concentrations above

150 lm (data not presented)

Fig 1 (A) Effect of NaCl on the conversion of AI (200 l M ) into AII

by ANCE Enzyme activity was measured using HPLC to quantify

the formation of AII in Hepes buffer (h, pH 7; n, pH 8) in the

pres-ence of NaCl (0–200 m M ) as described in Experimental procedures The enzyme activity is expressed as percentage of the maximum activity recorded at pH 8 in the absence of NaCl Values are the mean of three assays and the percentage standard error of the mean was 1–4% (B) Inhibition of ANCE and ACER by MKRSRGPSPRR Enzyme activity was determined using Abz-YRK(Dnp)P as described in Experimental procedures and is expressed as a percentage of the uninhibited activity.

Hydrolysis of BK and related peptides

Initial velocities for the hydrolysis of the BK peptides

were obtained by determining the rate of release of the

C-terminal dipeptide (Phe-Arg for BK, [Thr6]BK and

Ile-Ser-BK; Tyr-Arg for [Tyr8]BK) ANCE

consis-tently cleaved these peptides with much greater

effi-ciency (kcat⁄ Km) than ACER, mainly because of the

lower affinity of ACER for these substrates (Table 3)

In the case of ANCE, extending BK at the N-terminus

with Ile-Ser had no significant effect on the Km and

kcat, and replacing the Phe8 of BK with tyrosine

resul-ted in a modest increase in both the Km and kcat In

contrast, replacing Ser6 of BK with threonine resulted

in greatly increased affinity between the substrate and ANCE, but not ACER Indeed the Km value for the hydrolysis of [Thr6]BK was so low that it was difficult

to obtain accurate Km values using HPLC to quantify reaction rates at very low substrate concentrations We therefore used [Thr6]BK as an inhibitor of the hydro-lysis of Abz-YRK(Dnp)P and obtained a Ki value of

23 ± 4 nm, confirming the very high affinity displayed

by ANCE for this peptide

MKRSRGPSPRR is an invertebrate BK-like peptide predicted to be a cleavage product of a neuropeptide precursor gene in Aplysia californica [30] HPLC analy-sis showed that MKRSRGPSPRR was an excellent substrate for ANCE, but was resistant to hydrolysis

by ACER MS confirmed that reaction products were MKRSRGPSP ([M + H]+, m⁄ z 1014.3) and MKRSRGP ([M + H]+, m⁄ z 830.4), generated by the sequential cleavage of Arg-Arg and Ser-Pro MKRSRGPSPRR was a strong inhibitor of the hydro-lysis of Abz-YRK(Dnp)P with a Ki of 185 nm for the inhibition of ANCE (Fig 1B) In contrast, MKRSRGPSPRR, even at a concentration of 100 lm, did not significantly inhibit ACER activity, measured with the same fluorogenic substrate

Homology model of the structure of ACER

We generated a model of ACER based on the crys-tal structure of ANCE The homology model

Table 1 Effect of NaCl on the kinetic constants for the conversion of AI into AII by ANCE Kinetic constants for the conversion of AI into AII were determined as described in Experimental procedures and are expressed as the mean ± SEM (n ¼ 3).

Km(m M ) kcat(s)1) kcat⁄ K m (s)1Æl M )1) K

m (m M ) kcat(s)1) kcat⁄ K m (s)1Æ M )1)

Table 2 Effect of NaCl on the hydrolysis of peptides by ACER The

rate of hydrolysis of peptides (200 l M ) was determined in 0.1 M

Hepes ⁄ 10 l M ZnSO4, pH 7 as described in Experimental

proce-dures Values are mean ± SEM (n ¼ 3).

Substrate

Reaction rate (units ⁄ h)

[Leu5]Enkephalinamide b 10.1 ± 0.4 123.2 ± 1.8

a

Units of activity, nmol AII formed per lg ACER.bUnits of activity,

nmol dipeptide released per lg ACER.

Table 3 Kinetic constants for the hydrolysis of bradykinin-related peptides by ANCE and ACER –, No detectable hydrolysis of the peptide and no inhibition of the cleavage of Abz-YRK(Dnp)P by ACER.

Substrate

Km(l M ) kcat(s)1) kcat⁄ K m s)1(l M ))1 Km(l M ) kcat(s)1) kcat⁄ K m (s)1Æl M )1)

a Kmdetermined from the IC50value obtained by measuring initial rates of hydrolysis of the fluorogenic substrate Abz-YRK(Dnp)P (5 l M ) in the presence of different concentrations of MKRSRGPSPRR b Estimated from the initial velocity recorded at a substrate concentration 100 times greater than the K m

allowed us to compare the structure of the

sub-strate⁄ inhibitor binding sites between these related

enzymes, which are very similar in primary protein

structure, but display quite different enzymatic

prop-erties One of the striking differences between ANCE

and ACER predicted by our model is a significant

change in the electrostatic charge that lines the

sub-strate-binding channel, a change from predominantly

negative charges in ANCE to positive charges in

ACER (Fig 2) To gain insight into why BK

pep-tides bind with higher affinity to ANCE than to

ACER, we docked BK and [Thr6]BK into the

ANCE substrate channel The modelling predicts

that the negatively charged side chain of Asp360, as

well as Glu150, forms favourable ionic interactions

with the positively charged C-terminal arginine of

both substrates (Fig 3) Interestingly, in ACER, this

interaction is lost because Asp360 is replaced with

His368 (Table 4) The models of BK and [Thr6]BK

bound to ANCE suggest that the extra methyl group

of [Thr6]BK occupies a small hydrophobic pocket,

which is conserved in both ANCE and ACER The

models also suggest that the two peptides bind in a

similar orientation, with a b-turn centred on the

resi-dues Pro2-Pro3

Selective inhibitors of ANCE and ACER Inhibition constants were determined for RXPA380, RXPA381 and RXPA384 for both ANCE and ACER (Table 5) These values showed that RXPA384 was only slightly more potent as an inhibitor of ACER,

Fig 2 Surface representations of the elec-trostatic potential of ANCE and a homology model of ACER The proteins have been sliced in half to show the internal substrate-binding channel The N-chamber and C-chamber (N and C) are postulated to bind

up to
7 N-terminal residues and the C-ter-minal dipeptide of substrate, respectively Molecular surfaces and electrostatic poten-tial were calculated with the program SPOCK (http://quorum.tamu.edu) ANCE co-ordi-nates were obtained from the recently determined crystal structure (PDB accession code 1J36) The homology model of ACER was generated in SWISS - MODEL using the ANCE structure as a template Positive and negative charges are represented by shades

of blue and red, respectively, with neutral areas coloured white.

Fig 3 A stick diagram showing predicted electrostatic interactions between the C-terminal Arg9 of BK and ANCE The interactions between Asp360 of ANCE and the guanidino group of Arg9 of BK will be lost in ACER as Asp360 is replaced with His368.

whereas RXPA381 was able to distinguish between the

two enzymes with a selectivity factor of more than 100

in favour of ACER RXPA380 inhibited ACER with a

Kiof 4.8 lm, but did not inhibit ANCE, even at a

con-centration of 100 lm

To understand the molecular basis behind the

select-ive inhibition of ACER by RXPA380 and RXPA381,

these molecules were modelled into the binding sites of

ANCE and ACER The model of RXPA380⁄ ACER

shows that RXPA380 is bound in a very similar

orien-tation to the model generated for RXPA380⁄ C-domain

ACE [31] Phe1033 and Phe1103 of C-domain ACE

are important in forming a hydrophobic side of the S2¢

pocket for binding the tryptophan of RXPA380 Both

of these residues are conserved in ANCE, but in

ACER, Phe1103 is replaced with His519 (Table 4)

The other side of the S2¢ pocket is formed by two

adjacent valine residues in C-domain ACE (Val955

and Val956) Val955 is replaced by larger

phenylalan-ine and tyrosphenylalan-ine residues in ANCE and ACER,

respectively, which in our models are pointing away

from the inhibitor so that the change in the size of the side chain may have minimal effect on binding Val956

of C-domain ACE is conserved in ACER as Val372, but in ANCE this is replaced by Thr364, which redu-ces the hydrophobicity of the ANCE S1¢ pocket (Fig 4A) In ANCE, Gln266 with its large polar side chain replaces Ser275 and Thr858 of ACER and C-domain ACE, respectively (Table 4) In our model, the larger side chain of Gln266 restricts the space available and results in steric hindrance of the large indole ring of RXPA380 (Fig 4A)

In RXPA381, the P1¢ and P2¢ proline and trypto-phan residues of RXPA380 are replaced by smaller alanine residues The models of RXPA381 bound to

Table 4 Comparison of the residues that contribute to the S2¢

sub-site of human C-domain ACE (the residue numbers for human tACE

are in parentheses) with the N-domain of human ACE, ANCE and

ACER.

N-domain

ACE

C-domain

Table 5 Potency of RXPA series of compounds as inhibitors of

ANCE and ACER ANCE and ACER activities were measured using

the fluorogenic substrate Abz-YRK(Dnp)P (5 l M ) as described in

Experimental procedures –, No inhibition with 100 l M RXPA380.

Inhibitor

Ki(n M )

A

B

Fig 4 Representations of enzyme–inhibitor interactions (A) RXPA380 bound to C-domain ACE (grey), superimposed on the crystal structure of ANCE (yellow), highlighting differences between the proteins at the S 2 ¢ pocket The absence of inhibition of ANCE

by RXPA380 can be explained by the replacement of Thr858 with the larger Gln266, and Val956 with the polar Thr364 The combined effects of these changes will be to reduce the hydrophobic nature

of the S2¢ site and restrict the space available for the large indole ring of RXPA380 In ACER, the equivalent of Thr858 of C-domain ACE is the smaller Ser275, whereas Val956 is conserved as Val372 This is consistent with the inhibition of ACER by RXPA380 (B) Space-filling representation of RXPA381 bound to ANCE (left) and ACER (right) in the S2¢ pocket, comparing the differences in packing of the P 2 ¢ methyl group of RXPA381 (arrowhead) against Val372 of ACER or the equivalent Thr364 in ANCE The tyrosine and lysine residues interacting with the C-terminus of the inhibitor are labelled The figure was generated in PYMOL

ANCE and ACER show that the inhibitor is bound in

a similar orientation, but with variation in the

orienta-tion of the C-terminal residue (Fig 4B) All S1¢ and S2¢

residues interacting directly with RXPA381 are

con-served between ANCE and ACER except for the

aforementioned Val372 (ACER) and Thr364 (ANCE)

(Table 5) The molecular dynamic simulations suggest

that the methyl groups of the two alanines of

RXPA381 pack closely with Val372 of ACER, whereas

in ANCE, the methyl group of the terminal alanine

residue is orientated away from Thr364, reinforcing

the importance of the hydrophobicity of the valine side

chain

Discussion

We have characterized the effect of Cl– on ANCE

activity by determining the kinetic constants for the

hydrolysis of AI in the absence and presence of NaCl

(100 mm) The increased kcat⁄ Km observed at pH 7,

was entirely the result of a 3.5-fold lowering of the Km

for AI A similar level of enhancement was also

achieved in the absence of NaCl by changing the pH

conditions from 7 to 8, although in this case changes

in both the Km and kcat contributed to the increased

catalytic efficiency Although these effects are

signifi-cant, they are modest compared with the activation by

NaCl of the AI-converting activities of the C-domain

of human ACE [3,32] ACER hydrolyses AI extremely

slowly, an activity that is not stimulated by Cl–

Never-theless, a strong effect of NaCl on the peptidase

activity of ACER was observed when either [Leu5]

enkephalinamide or [Met5]enkephalinamide was the

substrate

Our observation that NaCl alters the affinity of

ANCE for AI suggests that the binding of Cl–induces

a conformational change in ANCE that influences the

hydrolysis of AI The molecular structures of two Cl–

-binding sites (Cl1 and Cl2) are known from the

struc-ture of human tACE [27], but no Cl– anions were

identified in the crystal structure of ANCE [28] The

Cl2 Cl–-binding site of tACE, 10 A˚ from the catalytic

zinc, is closer to the active site than Cl1 and comprises

the side chains of Arg522, Trp220 and Tyr224

Com-paring the structures of ANCE, ACER and tACE at

the Cl2 binding site suggests that ANCE and ACER

would not bind Cl–at the Cl2 site The substitution of

Pro519 in tACE by a glutamate in both ANCE and

ACER results in the carboxylic acid of this residue

residing in the space occupied by Cl–in the tACE

crys-tal structure [26]

The Cl1 binding site of tACE lies 20 A˚ from the

catalytic zinc and involves three contacts, Arg186,

Trp485 and Arg489 Whereas Arg489 is conserved, Arg186 and Trp485 of tACE are replaced by Tyr170 and Phe469 in ANCE It has been proposed that the Arg fi Tyr substitution may result in a Cl–-binding site more similar to the ACE Cl2 binding site [26] Although the Trpfi Phe substitution is expected to reduce the affinity for Cl–, it is possible that the Cl1 site in ANCE may still bind the anion and that this interaction is responsible for our observed increase in affinity of ANCE for AI In ANCE, the potential Cl1 binding site is adjacent to the peptide backbone of Lys495, which our modelling, together with recent site-directed mutagenesis studies on human ACE [33], suggest direct interactions between Lys495 and the C-terminus of the peptide substrate (Fig 3) The pres-ence of a Cl– ion at this site may have a stabilizing effect on binding certain substrates

In the N-domain of human ACE, and in ACER, the Cl1 site is altered by the replacement of Arg186 of tACE with His164 and His177, respectively, making it unlikely that Cl– will bind at this position in both these enzymes [26] However, there is a possibility that

an alternative Cl–-binding site exists in the N-domain

of human sACE, as the R500Q mutant of the human ACE N-domain, which removes the Cl2 site, responds

to 20 mm NaCl by a twofold increase in affinity for

AI [32] The strong NaCl-induced activation of ACER activity towards the amidated enkephalin substrates and the unlikely involvement of the Cl1 and Cl2 sites

in this effect suggest that a different anion site may also be present in ACER A similar proposal for a

Cl–-binding site, distinct from the two identified in tACE, has been put forward to explain the

Cl–-enhanced carboxypeptidase activity of human ACE2 [29] The lack of understanding of the molecu-lar mechanism by which Cl– influences the catalytic activity of ACEs is illustrated by the recent characteri-zation of ACE from the leech Theromyzon tessulatum [34] The residues forming both Cl1 and Cl2 in tACE are absolutely conserved in the leech enzyme, suggest-ing that this ACE would, like human C-domain, be strongly activated by NaCl However, the enzyme when expressed in mammalian cells responds with only modest activation (twofold) of the hydrolysis of Hip-His-Leu by NaCl with an optimal Cl– concentration

of 50 mm, and, thus, resembles the N-domain rather than the C-domain of human ACE

All the BK peptides used in this study were cleaved

by both ANCE and ACER, although ANCE was invariably the more efficient enzyme, displaying

kcat⁄ Kmvalues 30–100-fold greater than those obtained with ACER Our model of ANCE with either BK or [Thr6]BK docked in the substrate channel suggests

that the acidic side chains of Asp360, as well as

Glu150, form favourable ionic interactions with the

positively charged C-terminal Arg of the peptides

These residues are conserved in the human N-domain

and C-domain active sites (Table 5), both of which

efficiently cleave BK However, Asp360 of ANCE is

replaced with His368 in ACER, and this change in the

electrostatic charge in the S2¢ pocket is predicted to

reduce ionic interactions between ACER and the

guanidino group of the C-terminal arginine of the BK

peptides This may explain why the Kmvalues for the

hydrolysis of BK, Ile-Ser-BK, [Thr6]BK and [Tyr8]BK

by ACER are 20–75-fold higher than the

correspond-ing values for ANCE The model also suggests that an

N-terminal b-turn centred on the residues Pro2-Pro3

of BK and [Thr6]BK allows the peptides to fit tightly

into the larger (N chambers) of the two active-site

cav-ities, which may explain why BK peptides bind with

much higher affinity to ANCE and ACER than AI

BK adopts a similar conformation in models of BK

bound to human C-domain ACE (R J Bingham,

unpublished work), which would provide an

explan-ation for why BK is the physiological substrate that

displays the highest-affinity of any substrate of the

human enzyme [2]

The affinity of BK for ANCE is increased almost

fourfold by introducing an extra methyl group in

[Thr6]BK It has been shown previously that [Thr6]BK

has a markedly different solution structure to BK [35]

and has a greater tendency to adopt an N-terminal

b-turn, which was also a consistent feature of our

molecular modelling The dynamic structure difference

between BK and [Thr6]BK provides a possible

explan-ation for the difference in binding affinity of these two

BK peptides to ANCE

MKRSRGPSPRR is structurally related to

mamma-lian BKs and was shown to be an excellent ANCE

substrate In contrast, this peptide was resistant to

hydrolysis by ACER and did not compete with

sub-strate for the enzyme active site The surface of the

ACER active site is predicted to be positively charged,

which would present an unfavourable electrostatic

environment for Arg⁄ Lys-rich peptides attempting to

access the substrate-binding channel In contrast, the

negative charges lining the ANCE substrate

chan-nel would be expected to favour interactions with

positively charged peptide substrates, especially

MKRSRGPSPRR, which has positive charges along

the length of the peptide

RXPA380 (Cbz-Phew[PO2-CH]Pro-Trp-OH) is a

highly selective inhibitor of the C-domain of somatic

ACE, with the pseudo-proline and the tryptophan

resi-dues in the P1¢ and P2¢ positions of the inhibitor being

important for determining this selectivity [31] For both ANCE and ACER, it is clear that proline in the P1¢ position does not allow strong inhibitor–enzyme inter-action, as the substitution of the P1¢ proline of RXPA380 with alanine in RXPA384 (Cbz-Phew[PO2 -CH]Ala-Trp-OH) makes a much more potent inhibitor

of both ANCE and ACER The proline in RXPA380 probably restricts the orientation of the P2¢ side chain to

an orientation that is less favourable for interactions in the S2¢ pocket of ANCE Of the 12 residues of the S2¢ subsite of C-domain ACE that are predicted to interact with the RXPA380 in a model of the inhibitor–enzyme complex [31], only eight are strictly conserved in the N-domain, nine in ANCE and eight in ACER (Table 4) The adjacent valines (Val955 and Val956) that help form the S2¢ pocket of C-domain ACE appear to be involved in binding the tryptophan side chain of RXPA380 It has been proposed that replacement of these two residues in N-domain ACE with polar serine and threonine will limit favourable hydrophobic inter-actions between inhibitor and enzyme [31] RXPA380 inhibits ACER, albeit weakly, but not ANCE Our model of the ACER–RXPA380 complex shows the inhibitor bound in a very similar orientation to that des-cribed for C-domain ACE, with the side chain of Val372 (equivalent to Val956 of C-domain ACE) involved in ligand interaction at the S2¢ pocket The replacement of Val372 of ACER with the polar Thr364

in ANCE probably contributes towards the lack of inhibitory activity of RXPA380 This supports the hypothesis that the hydrophobicity of Val956 in C-domain ACE and Val372 in ACER is important for RXPA380 selectivity In our model, the larger side chain

of Gln266 restricts the space available for the large in-dole ring of RXPA380 and would therefore contribute together with Thr364 towards hindrance of RXPA380 binding to ANCE In contrast, Thr858 of C-domain ACE is replaced by the smaller Ser275, and ACE Val956 is conserved as Val372 in ACER, which is con-sistent with the inhibition of ACER by RXPA380 RXPA381, which has alanine in both the P1¢ and P2¢ positions, inhibits both ANCE and ACER, but displays 100-fold selectivity in favour of ACER This selecti-vity is consistent with the observation that RXP407 (Ac-Asp-Phew[PO2-CH]Ala-Ala-NH2) and Ac-Asp-Phew[PO2-CH]Ala-Ala-OH with a P1¢ and a P2¢ alanine are also selective inhibitors of ACER [17] The side chain of Gln266 of ANCE, which forms the back of the

S2¢ site, is too distal (8 A˚) to interact with the P2¢ side chains of RXPA381 and RXP407, and therefore will not influence the binding of these less bulky inhibitors The unexpected result that ACER is inhibited by both an N-domain-selective and a C-domain-selective

inhibitor demonstrates the dangers of classifying ACEs

as either N-domain-like or C-domain-like Molecular

models of inhibitors complexed with ANCE and ACER

have suggested structural explanations for these

obser-vations and provided new insights into how structural

diversity in the ACE substrate channel can lead to

important differences in enzymatic properties In

addi-tion, our models of BK docked at the ACE active site

have provided an explanation for the evolutionarily

conserved tight binding of this substrate to ACE

Experimental procedures

Enzyme substrates and inhibitors

Peptides were purchased from Sigma-Aldrich (Poole,

Dorset, UK) RXPA380 (Cbz-Phew[PO2-CH]Pro-Trp-OH),

RXPA381 (Cbz-Phew[PO2-CH]Ala-Ala-OH), RXPA384

(Cbz-Phew[PO2-CH]Ala-Ala-OH) were synthesized as

des-cribed previously [8,31] Abz-YRK(Dnp)P was a gift

from Professor Adriana K Carmona, Department of

Bio-physics, Division of Nephrology, Escola Paulista de

Medici-na, Universidade Federal de Sao Paulo, Sao Paulo, Brazil

Expression and purification of recombinant ANCE

and ACER

Recombinant ANCE and ACER were produced by

expres-sion in Pichia pastoris, as described previously [20,25]

Secre-ted ANCE and ACER were purified to homogeneity from

the culture medium by using a combination of hydrophobic

interaction and ion-exchange chromatography (NH4)2SO4

was added to the culture media to a final concentration of

1.5 m, and, after centrifugation and filtration (0.2 lm pore

size; Minisart, Sartorius Ltd, Epsom, Surrey, UK), the

cul-ture media were applied to a column (12 cm· 2.6 cm)

packed with Phenyl-Sepharose Fast Flow 6 (Amersham

Biosciences, Chalfont St Giles, Buckinghamshire, UK)

pre-equilibrated with 1.5 m (NH4)2SO4⁄ 20 mm Tris ⁄ HCl,

pH 8.0 Protein was eluted with a decreasing gradient of

(NH4)2SO4(1.5–0 m; over 500 mL; flow rate of 5 mLÆmin)1)

and monitored using a UV detector set at 280 nm

Protein-containing fractions were pooled and dialysed against

20 mm Tris⁄ HCl, pH 8.0, before being applied to an

ion-exchange column (HiTrap Q HP, 5 mL bed volume;

Amer-sham Biosciences) Protein was eluted using a 200 mL

gradi-ent of increasing concgradi-entration of NaCl (0–1 m), at a flow

rate of 5 mLÆmin)1 Fractions containing enzyme activity,

determined using Hip-His-Leu as the substrate [36],

were pooled and dialysed against 100 mm Tris⁄ HCl

(pH 7.0)⁄ 50 mm NaCl ⁄ 10 lm ZnCl2, before being

concen-trated to 1 mg protein per ml of buffer using a centrifugal

concentrator (Microsep 10k; Pall Life Sciences, Portsmouth,

Hampshire, UK) The final protein concentration was

determined by absorbance at 280 nm Cl–-free protein was produced by dialysing 1 mL protein solution (1 mgÆmL)1) against 5 L MilliQ water for 24 h followed by dialysis against 100 mm Hepes (pH 8.0)⁄ 10 lm ZnSO4for 24 h

Enzyme assays Dipeptidyl carboxypeptidase activity towards peptide sub-strates was determined by HPLC quantification (214 nm) of the reaction products (AII for the hydrolysis of AI; Phe-Arg for the hydrolysis of BK, Ile-Ser-BK and [Thr6]BK; Tyr-Arg for the hydrolysis of [Tyr8]BK; MKRSRGPSP for the hydrolysis of MKRSRGPSPRR; Tyr-Gly-Gly, Phe-Leu-amide and Met-Leu-Phe-Leu-amide for [Leu5]enkephalinPhe-Leu-amide and [Met5]enkephalinamide; Tyr-Gly-Gly, Phe-Leu and Met-Leu for [Met-Leu5]enkephalin and [Met5]enkephalin) Unless otherwise stated, the reactions were carried out at 35
C in

100 mm Hepes (pH 8.0)⁄ 50 mm NaCl ⁄ 10 lm ZnSO4 in a final volume of 20 lL for AI and larger volumes (200 lL

to 1 mL) for BK and BK-related peptides Reactions were stopped by either addition of trifluoroacetic acid to a final concentration of 2.5% or, for larger volumes, immersion in boiling water for 5 min HPLC analysis required different reverse-phase columns and elution conditions to achieve peptide separation The products of AI, MKRSRGPSPRR, and BK hydrolysis were resolved using a Phenomenex Jupiter C18 (5 lm particles, 250· 4.6 mm; Phenomenex, Macclesfield, Cheshire, UK) column, whereas the separation

of BK 1–5 and BK 1–7 required a SuperPac Pep-S column (5 lm particles, 250 mm· 4 mm; Amersham Biosciences) The following elution gradients of acetonitrile in 0.1% tri-fluoroacetic acid at a flow rate of 1 mLÆmin)1 were used: 15–36% acetonitrile over 14 min for AII; 6–24% acetonitrile over 22 min for Phe-Arg and MKRSRGPSP; 6–18% aceto-nitrile for BK 1–5 and BK 1–7 over 20 min; 0–24% acetonit-rile over 20 min for the separation of Tyr-Gly-Gly, Phe-Leu, Met-Leu, Phe-Leu-amide and Met-Leu-amide Identification

of peptides by MS was performed using a Q-Tof MS⁄ MS instrument Hip-His-Leu hydrolysis was assayed as des-cribed previously [36]

The kinetics of inhibition of ANCE and ACER by BK, BK-related peptides and phosphinic acid inhibitors were determined by measuring the effects on initial rates of hydrolysis of Abz-YRK(Dnp)P (5 lm) in 100 mm Hepes,

pH 8.0, 50 mm NaCl and 10 lm ZnSO4(final reaction

Abz-YRK(Dnp)P, a fluorogenic substrate based on the structure

of N-acetylSDKP [7], with Kmvalues of 6.64 ± 1.1 lm and 4.60 ± 1.4 lm, respectively The reactions were performed

at 20
C in 96-well black plastic plates (Corning Life Sciences, High Wycombe, Buckinghamshire, UK) using a Victor2 fluorimeter (PerkinElmerTM, Turku, Finland) to quantify the rate of increase in fluorescence (kem 430 nm and kex 340 nm) The reaction was started by adding the

substrate to the enzyme in 100 mm Tris⁄ HCl

(pH 7.0)⁄ 100 mm NaCl ⁄ 10 lm ZnCl2.

Kinetic parameters and IC50values were calculated using

nonlinear regression curve-fitting programs (figp; Biosoft,

Cambridge, UK) Error values are standard deviations of

the parameters calculated from the fitted curve by figp The

Kiof inhibition of ANCE by [Thr6]BK was determined by

measuring the kinetics of Abz-YRK(Dnp)P hydrolysis in

the presence of 0, 10, 20, 50 and 80 nm [Thr6]BK

Molecular modelling

The model of D melanogaster ACER was generated in

swiss-model [37] using the first approach mode and the

crys-tal structure of ANCE as a template (Protein DataBank

accession code 1J36) The zinc atom was manually

posi-tioned, co-ordinated by His375, Glu376 and His379, which

were deduced to be the co-ordinating residues by sequence

alignment The co-ordinates of BK and [Thr6]BK were

gen-erated in pymol (http://www.pymol.org), and manually

posi-tioned into the binding channel of ANCE and ACER using

the molecular visualization program O The peptide was

aligned such that the carboxy group of the scissile peptide

bond was orientated towards the zinc according to the

pro-posed catalytic mechanism [27] The large N-chamber and

C-chamber readily allowed positioning of the peptide with

minimal steric clashes The model was then solvated with

explicit water molecules in a 20 A˚ sphere centred on the

pep-tide This model was improved by energy minimization and

molecular-dynamics simulations using the ds Modelling

soft-ware (Accelrys, San Diego, CA, USA) All energy

calcula-tions were performed using the CHARM22 force field, and

were restricted to the 20 A˚ sphere centred on the peptide

The nonbonded cut-off was set to 12 A˚ Initial optimization

was performed by two stages of energy minimization, firstly

500 steps of a conjugate gradient minimization, followed by

1000 steps using the adopted basis Newton–Raphson

algo-rithm This was followed by heating to and equilibrium at

300 K before a 1000-step molecular-dynamics simulation

with time steps of 0.001 ps Co-ordinates of RXPA380 were

kindly provided by Philippe Cuniasse, Departement d’Etudes

et d’Ingenierie des Proteines, Commissariat a l’Energie

Atomique, CE-Saclay, Gif-Sur-Yvette, France Co-ordinates

of RXPA381 were generated in pymol These co-ordinates

were then superimposed on to ANCE and ACER assuming a

similar binding orientation to ACE C-domain This model

was then solvated with explicit water molecules in a 20 A˚

sphere centred on the peptide and then subjected to the

molecular modelling scheme described above

Acknowledgements

We thank Adriana K Carmona (Universidade Federal

de Sao Paulo) for ACE substrates and Pam Gaunt

(University of Leeds) for technical expertise, Alison Ashcroft (University of Leeds) for mass spectrometry, Philippe Cuniasse (Commissariat a l’Energie Atomi-que, CE-Saclay) for the pdb file of RXPA380, and Pierre Corvol, Tracy Williams and Xavier Houard (College de France, Paris) for Pichia expressing ANCE and ACER We acknowledge the support of the Bio-technology and Biological Sciences Research Council through a studentship to R.J.B and a grant to A.D.S and R.E.I (No 89⁄ S19378)

References

1 Erdos EG (1990) Angiotensin-I converting enzyme and the changes in our concepts through the years Hyper-tension 16, 363–370

2 Corvol P, Eyries M & Soubrier F (2004) Peptidyl-dipep-tidase A⁄ angiotensin I-converting enzyme Handbook of Proteolytic Enzymes(Barrett, AJ, Rawlings, ND & Woessner, JF, eds), pp 332–346 Elsevier⁄ Academic Press, Amsterdam

3 Wei L, Alhencgelas F, Corvol P & Clauser E (1991) The 2 homologous domains of human angiotensin-I-converting enzyme are both catalytically active J Biol Chem 266, 9002–9008

4 Wei L, Clauser E, Alhencgelas F & Corvol P (1992) The 2 homologous domains of human angiotensin-I-converting enzyme interact differently with competitive inhibitors J Biol Chem 267, 13398–13405

5 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

6 Rousseau A, Michaud AM-TC, 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-converting enzyme J Biol Chem 270, 3656–3661

7 Araujo MC, Melo RL, Cesari MH, Juliano MA, Juliano L & Carmona AK (2000) Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme Biochemistry 39, 8519–8525

8 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

9 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