Table 1 Catalytic zinc sites (Zn–L1,L2,L3,L4,L5) of representative Zn2þenzymes (referred fromref. 10).
L1 L2 Zn2+
L3 L4
k A A mA A
L5
AA = am in o aci ds nA A
Enzymes L1 k L2 m L3 n L4 L5 References
Carboxypeptidase (CPD) Family
Bovine A His 2 Glu2b 123 His(C) H2O 19,20
Bovine B His 2 Glu 123 His H2O 21
Rat A2 His 2 Glu 123 His H2O 22
Human A2 His 2 Glu2b 124 His H2O 23
Avian D His 2 Glu2b 103 His H2O 24
Bovine CPD A His 2 Glu2b 123 His H2O 25
Human CPD A2 His 2 Glu 123 His H2O 26
Thermolysin Family
Bacillus His 3 His 19 Glu(C) H2O 27
thermoproteolyticus
Pseudomonas His 3 His 19 Glu H2O 28
aeruginosa
Staphylococcas His 3 His 19 Glu H2O 29
aureus
Human leucotriene His 3 His 18 Glu(C) H2O 30
A4hydrolase
Human neutral His 3 His 58 Glu(C) H2O 31
endoprotease
Clostridium His 3 His 34 GluL(C) H2O 32,33
botulinum neurotoxin A
Streptomyces His 6 His 35 His(C) H2O 34
albusG DD-CPD
D-Ala-D-Ala carboxy- His 6 His 60 His(C) H2O 35
peptidase (VanX)
Astacin Superfamily His 3 His 5 His H2O Tyr 36,37
Serratia Family
Pseudomonas His 3 His 5 His(C) H2O Tyr 38,39
aeruginosa alkaline protease
Serratia marcescens His 3 His 5 His H2O Tyr 40
Metalloprotease
Snake Venom Protease Family
Crotalus adamanteus His 3 His 5 His(C) H2O 41,42
(oradamalysin II)
Human TNF-- His 3 His 5 His H2O 43
converting enzyme (TACE)
MMP Family
Human fibroblast His 3 His 5 His(C) H2O 44
collagenase (MMP-1)
Human fibroblast His 3 His 5 His H2O 45,46
collagenase (MMP-1)
Human matrilysin His 3 His 5 His H2O 47
(MMP-7)
Human stromelysin-1 His 3 His 5 His H2O 48–50
(MMP-3)
Human stromelysin-1 His 3 His 5 His H2O 51,52
(MMP-3)
8.23.3.1 Carboxypeptidase Family
The carboxypeptidase family of exopeptidases and the thermolysin family of endopeptidases are the most studied examples of carboxylate-assisted Zn2þ-bound watercatalysis.4,8,11,90,91
The Zn2þ-containing bovine pancreatic carboxypeptidase A and B (CPDA and B) were two of the earliest identified Zn2þmetalloenzymes. They catalyze the degradation of food proteins leading to amino acids such as Phe, Trp, Lys, and Arg, complementing the actions of chymotrypsin, pepsin, and trypsin. X-ray crystal structures of CPDA-inhibitor complexes, spectroscopic studies of inhibitor and substrate binding of CoII-substituted enzymes,89,97and XAFS studies of the effect of pH and inhibitorbinding on the Zn2þcoordination sphere98have provided evidence for a mechanism invol- ving Zn2þ–OH2and a general acid/base role for Glu270, assisted by the guanidinium of Arg127 for the anionic transition state. A Glu carboxylate similarly activates Zn2þ–OH2in thermolysin.91
Table 1 continued
Enzymes L1 k L2 m L3 n L4 L5 References
Human progelatinase His 3 His 5 His(C) 200 Cys(N) 53
(MMP-2)
Human collagenase-3 His 3 His 5 His H2O 54,55
(MMP-13)
Murine adenosine His 1 His 196 His(C) H2O 56,57
E. colicytidine Cys 2 Cysb 26 Hisb(N) H2O 58,59
deaminase
E. colipeptide His 3 His 41 Cys(N) H2O 60–62
deformylase
E. colicolicin Hisa 3 His 24 His(N) H2O 63
E7 DNAse
Human GTP Cys 2 His2b 41 Cysa(C) H2O 64
cyclohydrolase I
Bacteriophage Cys2b 7 His 104 His(N) H2O 65
T7 lysozome -Lactamase Family
Bacillus cereus His2 1 His 60 His(C) H2O 66–69
Pseudomonas His2 1 His 59 His(C) H2O 70
aeruginosa
E. colialkaline Asp 3 His 80 His(C) H2O 71,72
phosphatase
Bacillus cereus Glu 3 His 13 His(N) H2O 73
phospholipase C
Penicillum citrinum Aspa 3 His 12 His(N) H2O 74
P1 nuclease
Hepatitis C Cys 1 Cys 45 Cys(C) H2O 75,76
virus proteinase CA Family
Homo Sapiens CA I His 1 His 22 His(C) H2O 77
Homo Sapiens CA II His 1 His 22 His(C) H2O 78
Bovine CA III His 1 His 22 His(C) H2O 79
Murine CA IV His 1 His 22 His(C) H2O 80
Rat 6-pyruvoyl- His 1 His 24 His(N) H2O 81,82
tetrahydropterin synthase
E. coli5-amino- Cysa 1 Cys 7 Cys(N) H2O GluL 83
aevulinate dehydratase
E. colifuculose-1- His 1 His 60 Hisb(C) H2O GluL 84–86
phosphate aldolase
E. colifructose- His 115 His 38 Hisb H2O 87,88
1,6-bisphosphate aldolase
a The numberof amino acids as a spacerbetween ligands L1 and L2 isk, that between L2 and L3 ism, and that between L3 and L4 isn.
The symbols N and C indicate that L3 is located on the amino(N) or the carboxy (C) side of L2, respectively. The subscripts,, refer to the- or 310helix and-sheet structure which supplies the ligand. The letter subscript L is an amino-acid sequence of<5 residues between two structural elements. The subscripts a and b indicate the ligand is either one (or two, 2) residues after or before the secondary structural element.
Scheme 2shows the proposed mechanism of CPDA.11,90 The catalytic Zn2þ site of CPDA is comprised of His69, Glu72, His196, and a water molecule. The first two ligands are separated by a short spacer of two residues in a seven-amino-acid loop region between a-sheet and/or-helix, while His196 is the last residue in a-pleated sheet extending from amino acids 191 and 196. This site is highly conserved throughout the extended CPD family. In the precatalytic Michaelis complex with the substrate, the carbonyl oxygen is hydrogen-bonded to Arg127 to assist nucleo- philic attack by the Zn2þ-bound H2O (orOH) activated by Glu270 (Scheme 2a). The anionic tetrahedral transition state is stabilized by chelation to Zn2þand interaction with Arg127, and protonated by Glu270 (Scheme 2b) to give the CPDA-product complex (Scheme 2c). The inter- actions of the substrate with Arg145, Arg127, and the carbonyl oxygen of Ser197 were confirmed by X-ray crystal structure analysis.99,100 A modified mechanism has been proposed, which includes interactions of the nitrogen of the scissile peptide bond with Glu270 and Tyr248, as shown inScheme 2dand 2e.91
8.23.3.2 Thermolysin Family
The structure of the active site of thermolysins is similar to that of carboxypeptidases, although the amino-acid spacers and the overall tertiary structures of these two enzymes are different.91The Zn2þ ion in thermolysin from Bacillus thermoproteolyticus is coordinated by His142, His146, Glu166, and a water molecule in a tetrahedral geometry (Scheme 3). The position of Glu143 is almost superimposable on Glu270 of CPDA (Scheme 2), suggesting that Glu143 similarly func- tions as the general acid/base in catalysis and that the chemical and stereochemical combination
Carboxypeptidase A
(CPDA) Thermolysin β-Lactamase
(Bacillus cereus)
Matrix metalloproteinase (Human fibloblast collagenase)
(MMP)
Astacin Carbonic anhydrase (CA) (Bovine CA III)
Scheme 1
of the Zn2þion and these two Glu CO2(Glu143 of thermolysin and Glu270 of CPDA) is crucial to the hydrolytic activities (Schemes 3a–3c). His231 of thermolysin and Tyr248 of CPDA are considered to be proton donors.91However, the replacement of Tyr248 of CPDA had little effect on enzymatic activity, suggesting that Tyr248 is not always required for substrate hydrolysis.
Comparison of the ligands to Zn2þ in human leukotriene A4 hydrolase101,102 with those in thermolysin allowed its classification as a thermolysin type (Table 1). Although the hydrolase reaction involved is different (Scheme 4), nucleophilic attack at the substrate by Zn2þ–OH2 is a common feature.
VanX is a carboxypeptidase that hydrolyzes the amide bond of a dipeptide, D-alanyl-D-alanine (D-Ala-D-Ala).35,103 This enzyme is essential for vancomycin-resistant enterococci (VRE), because it produces peptidoglycan precursors terminating in D-alanyl-D-lactate (D-Ala-D-Lac) in place of D-Ala-D-Ala, resulting in a 100-fold decrease in the affinity with vancomycin.104,105Scheme 5depicts a proposed mechanism of VanX, where the active-site structure is similarto those of CPDA and thermolysin. The Zn2þ-bound H2O activated by Glu181 attacks
(a)
(b)
(c)
(d)
(e) Hydrophobic
Scheme 2
the carbonyl carbon polarized by zinc(II) (Scheme 5a) to give a tetrahedral intermediate, which is stabilized by Zn2þ and Arg71 (Scheme 5b). Glu181 gives the proton to the nitrogen that is hydrogen bonded to the carbonyl oxygen of Tyr109 (Scheme 5c) pr ior to the cleavage of the peptide bond (Scheme 5d).
Angiotensin-converting enzyme (ACE) is also a zinc(II) dipeptidase, responsible for the forma- tion of an octapeptide angiotensin II (Asp–Arg–Val–Tyr–Ile–His–Pro–Phe) by hydrolyzing the dipeptide (His-Leu) at the C-terminus of a decapeptide, angiotensin I (Asp–Arg–Val–Tyr–Ile–His–
Pro–Phe–His–Leu).106,107ACE is a key enzyme for the rennin–angiotensin blood-pressure-control- ing system. Although the crystal structure of ACE has not as yet been determined, the DNA sequence of ACE is homologous to that of thermolysin.107
(a)
(d)
(b)
(c)
Scheme 3
arachidonic acid
leukotriene A4
leukotriene A4 hydrolase
leukotriene B4 Scheme 4
8.23.3.3 Astacin Superfamily
This superfamily is named after Astacus protease, or astacin, which was identified as a Zn2þ protease in 1988.108 The consensus Zn2þ-binding site amino-acid sequence within astacin is HExxHxxGxxH, which also was found in some metalloproteinases; these were divided into four subclasses, comprising 33 proteases, in this superfamily.109 Representative structures of enzymes from the four common subfamilies were characterized, i.e., (i) serratia family; (ii) snakevenom protease family; (iii) MMP family; and (iv) -lactamase family. The tumornecrosis factor -converting enzyme (TACE) forms a member of the second subgroup.
In contrast to the structure of CPDA or thermolysin, the Zn2þ ion in serratia has the fifth ligand, Tyr149, as shown inScheme 6. The Zn2þ–O(Tyr149) length is 2.75 A˚ in the solid state.110–112 Since Tyr149 is located in a similar position to His231 of thermolysin (Scheme 3), it is considered that these two amino-acid residues have a similar function. Glu93 in astacin may work to shuttle protons between the Zn2þ-bound waterand the nitrogen atom of the scissile peptide bond (Schemes 6b–6d) in analogy to Glu270 of CPDA and Glu143 of thermolysin.
8.23.3.3.1 MMP family
MMP are structurally similar to the serratia family but without Tyr at the Zn2þ active center.
They mediate the breakdown of the extracellular matrix, which is associated with normal tissue remodeling processes during pregnancy, wound healing, and angiogenesis.113–116 Therefore, MMPs are potential targets for therapeutic drugs against inflammatory, malignant, and degen- erative diseases.117
A proposed mechanism for MMP catalysis is depicted in Scheme 7.117,118 The Zn2þ ion is coordinated by three His(218, 222, and 228) and one water molecule. A Glu carboxylate
(a) (b)
(d) (c)
Scheme 5
(e.g., Glu219 in MMP-1 (fibroblast collagenase) or Glu198 in MMP-8 (neutrophil collagenase)) acts as a base to activate Zn2þ-bound water(Scheme 7a) like Glu270 in CPDA, Glu143 in thermolysin, and Glu93 in astacin. It is proposed that the scissile amide carbonyl oxygen coordinates to the Zn2þ ion. A proton transferred from Zn2þ-bound waterto Glu219 is again shuttled onto the nitrogen atom of the scissile amide (Scheme 7b) that is then cleaved (Scheme 7c). A conserved Ala182 stabilizes positive charge at the nitrogen of the scissile amide.
E. coli cytidine deaminase (CDA) is an enzyme that catalyzes hydrolytic deamination of cytidine to uridine (or from 20-deoxycytidine to 20-deoxyuridine) (Scheme 8).119,120 This enzyme is considered to be a member of the MMP family, although the Zn2þ-binding site contains two Cys and one His residues. On the basis of the X-ray crystal structure of a transition-state analogue complex,58,59a mechanism was proposed as shown inScheme 8, in which Glu104 serves multiple roles: the deprotonation of the Zn2þ-bound waterand simultaneous protonation to N3 of the substrate (Scheme 8a), stabilization of the first tetrahedral transition state (Scheme 8b), and protonation of the leaving amino group at the 4-position of the substrate in the second tetra- hedral intermediate (Scheme 8c).119,120 Upon elimination of NH3, an E–P complex is formed (Scheme 8d) and the enzyme goes into the next turnover. The deamination mechanism ofE. coli
(a)
(b)
(d)
(c)
(e) 2.75A°
Scheme 6
adenosine deaminase (ADA) is considered to be similar.10 However, the Zn2þ-binding site of ADA contains three His residues. The difference in Zn2þligands and tertiary structures of ADA and CDA led to the proposal that the common features in the transition-state stabilization arose from convergent evolution.58
8.23.3.3.2 b-Lactamase family
-Lactamases are bacterial enzymes, some of which play a crucial role in the resistance of pathogens to -lactam antibiotics. They are grouped into four classes (A, B, C, and D).121–126 Classes A, C, and D are serine enzymes using serine as a nucleophile,125which are excluded in this text. Class B -lactamases include mononuclearzinc(II)--lactamases127–131 and dinuclear zinc(II)--lactamases.131–134 A Zn2þ-containing -lactamase II hydrolyzes the -lactam ring of a variety of penicillins (e.g., benzylpenicillin) and cephalosporins. At pH 7 and 30C, the half- life of benzylpenicillin bound to -lactamase II is ca. 0.5 ms. The first structure of the B. cereus enzyme showed one Zn2þ coordinated by His86, His88, and His149, and a water in a tetrahedral arrangement.66,67 Scheme 9 represents a proposed mechanism for the monozinc(II)- -lactamase reaction with benzylpenicillin.130 Two pKa values, pKa1 and pKa2, of -lactamase were found to be 5.6 and were assigned to a Zn2þ-bound water(Scheme 9a and 9b), and Asp90 carboxylic acid (Schemes 9c and 9d). The mechanism involves the binding of the substrate carbonyl to Zn2þ (Scheme 9c), nucleophilic attack of the Zn2þ-bound HO to the carbonyl carbon of the substrate (Scheme 9c), and subsequent stabilization of the oxyanion by Zn2þ (Schemes 9d and 9e).124,125,130
Alkaline phosphatase (AP) from Escherichia coli is a homodimeric, nonspecific phospho- monoesterase consisting of 449 amino acids, two Zn2þions, and one Mg2þion (Table 1).135–142 One Zn2þ(Zn2þ(1) in Scheme 10) is coordinated by two His and one Asp and is proposed to provide the catalytically required Zn2þ–OH2. The E. coli AP is a prototype for all alkaline phosphatases (especially the mammalian phosphatases) and is functionally related to the phos- phoesterases, such as phospholipase C from Bacillus cereus73 and P1 nuclease from Penicillum citrinum.74 Scheme 10 shows a proposed mechanism for AP.143 In Schemes 10a and 10b, the formation of an AP–ROPO32complex involves coordination of a phosphate oxygen to Zn2þ(1) and additional interactions between the RO group of the substrate and Zn2þ(2), as well as hydrogen bonds to the guanidinium group of Arg166. The Zn2þ(2)–bound Ser102 occupies the
(a)
(b) (c)
Scheme 7
position opposite to the leaving OR group (Schemes 10b and 10c). By nucleophilic attack of Ser102 on ROPO32
, the POR bond is cleaved and the AP–PO42
complex (E–P intermediate) is formed, in which PO42 is stabilized by Arg166. At alkaline pH, the Zn2þ–bound water (or hydroxide) attacks the covalent E–P intermediate forming the noncovalent EPO43
(orEHPO42) complex (Scheme 10cand10d). Recently, a revised mechanism involving Mg2þ was proposed.142
8.23.3.4 CA Family
CA is the well-studied classic Zn2þ enzyme.92–96 CA catalyzes the reversible formation of bicarbonate and a hydrogen ion from water and carbon dioxide. So far, seven human CA isozymes have been identified: CA I–VII, which are expressed in varying amounts in different tissues and cell locations. All the isozymes have at least 31% amino-acid homology and CA I–III share 58–60% amino-acid identity.
cytidine (C)
H2O N+H4 cytidine deaminase
uridine (U)
(a)
(b)
(d)
(c) 3
4
3 4
E-P complex NH3
first tetrahedral intermediate second tetrahedral intermediate Scheme 8
In general, the catalytic unit, Zn(His)3, is buried in a hydrophobic cavity. Three His moieties are hydrogen-bonded to the amide oxygen (side chain) of Gln92, the amide oxygen of the Asn244 backbone, and the Glu117 carboxylate, respectively (Scheme 11).11,143 The deprotonation of a Zn2þ-bound H2O (Scheme 11a) to a distinct Zn2þ-bound OH (Scheme 11b) occurs with a pKa value of 6.9, which may be facilitated by the base-relaying of Glu106 and Thr199. The Zn2þ-bound OHattacks CO2(Scheme 11c) that is bound in a hydrophobic pocket constructed by Val143, Val121, Trp209, and Leu198, to yield HCO3 ion, stabilized by coordination to Zn2þ(Scheme 11d). Upon release of HCO3
, the Zn2þ-bound H2O is regenerated.
Aldolases II are lyases that catalyze reversible aldol reactions and yet are classified in the CA family because of theircommon amino-acid ligands (Table 1). Aldolases consist of two types.
The type-I aldolases, which primarily are found in animals and higher plants, activate donor substrates, typically dihydroxyacetone phosphate (DHAP), via formation of a Schiff base with a lysine side chain as an intermediate (Schemes 12aand12b). The type-II aldolases, which occurin yeasts andEscherichia coli, utilize the zinc(II) ion144–146to polarize the carbonyl group (Schemes 12c and 12e). The X-ray crystal structure of the type-II fuculose-1-phosphate (Fuc–1-P) aldolase revealed that the zinc(II) ion is coordinated by three imidazole groups of His(92, 94, 155) and one carboxylate of Glu73. On the basis of the X-ray crystal structure of Fuc–1-P aldolase complexed with phosphoglycolohydroxamate (PGH), which is a substrate analogue and an efficient inhibitor, a role of the zinc(II) ion was proposed in which it stabilizes the enediolate–DHAP intermediate. All the observations have also suggested a role for zinc(II) in polarizing the
(a) pKa=5.6
pKa=5.6 (b)
–H+
(d) (c)
(e) (f)
Benzylpenicillin
Scheme 9
carbonyl bond to increase the acidity of the-methylene protons, so as to be readily abstracted by the carboxylate of Glu73 (Scheme 12c).
8.23.3.5 Other Zinc(II) Hydrolases 8.23.3.5.1 Histone deacetylase (HDAC)
Histone deacetylase (HDAC) is an enzyme that hydrolyzes acetylated lysines in histone proteins to change the conformation of the nucleosome (a complex of histone protein with nucleus DNA) and hence regulates gene expression.147–150 The X-ray crystal structure of a HDAC homologue complexed with a specific inhibitorrevealed that a Zn2þion in the active site is coordinated by Asp168, His170, Asp258, and a water molecule in a tetrahedral geometry. With the presence of an Asp–His charge-relay system, a mechanism is proposed for the deacetylation reaction as shown inScheme 13.151
8.23.3.5.2 Anthraxlethal factor
The lethal factor (LF) of anthrax, which is responsible for the pathogenesis of anthrax, was found to possess highly specific protease acitivities against mitogen-activated protein kinase kinases (MAPKKs) family, leading to inhibition of one ormore signaling pathways.152,153The X-ray crystal-structure analysis revealed that the anthrax LF comprises four domains: domain I
(a) (b)
(d) (c)
HPO42– HPO42–
ROPO32–
RO–
Scheme 10
binds the membrane-translocating component of anthrax toxin; domains II, III, and IV make a deep groove that captures the substrate, the N-terminal sequence of MAPKK-2.154 Among these four domains, the domain IV possesses Zn2þmetalloproteinase activity. The Zn2þion is coordinated by His686, His 690, Glu735, and a watermolecule, and resembles the active Zn2þsite of carboxy- peptidases (Scheme 14). Glu687 from the HExxH motif is located 3.5 A˚ from the Zn2þ-bound water, implying that it functions as a general base to activate the Zn2þ-bound water. The hydroxy group of Tyr728 hydrogen bonds to the Zn2þ-bound water from the opposite side of Glu687. Tyr728 possibly acts as a general acid to activate the amino group of the substrate.
Moreover, the crystal structure of the complex of the anthrax LF with an intact natural substrate (Met–Leu–Ala–Arg–Arg–Lys–Pro–Val–Leu–Pro–Ala–Leu–Thr–Ile–Asn–Pro) was solved (Scheme 14), where the main-chain peptide bond between Ala11 and Leu12 is to be cleaved. The amino-acid residue nearest to the Zn2þ ion was Pro10, about 6 A˚ away from the Zn2þ-bound water, a fact suggesting that this structure represents a ‘‘precleavage’’ complex.
8.23.4 DEVELOPMENT OF Zn2þHYDROLASE INHIBITORS
Specific inhibitors for Zn2þhydrolases have been very actively developed, mostly on the basis of the acquired information on the structures of the active centers. They are extremely helpful in
(a) (b)
(d) (c)
HCO3– H2O
CO2 PKa=6.9
Scheme 11
understanding more detailed mechanisms and, moreover, they may be good drugs against diseases involving Zn2þenzymes.155–159
8.23.4.1 Carboxypeptidase Inhibitors
The commercially most successful zinc(II) carboxylase inhibitors include captopril (1), enalapril (2a), enalaprilat (2b) (the diacid metabolite of 2a), and trandolapril (3).155–158,160–162 Forstruc- tures of these drugs, seeScheme 15. They all act against the ACE that converts angiotensin I to angiotensin II. These compounds have been used for years as successful orally active drugs to reduce high blood pressure by decreasing the ACE activities that play a major role in the
(a) (b)
G3P
Type I aldolase (animals, plants) (via enamine)
Type II aldolase (yeast) (via Zn2+–enolate) (c)
R=CH2OH, CH3
Zn2+
(d)
L-lactoaldehyde (e) DHAP
G3P
Scheme 12
renin–angiotensin blood regulating system (the half-inhibitory concentrations (IC50) are marked in Scheme 16). It is remarkable that these drugs were developed without detailed structural knowledge about ACE, and the discovery of the original ACE inhibitor captopril (1) by Ondetti et al. is a legendary story.160–162 The interaction of the substrate (angiotensin I) and the
Scheme 13
scissile bond
The substrate (MAPKK tail)
S1 pocket 2.6 Å
ca.6 Å
Scheme 14
inhibitors with the active site of ACE was hypothesized to be as shown in Scheme 16. The original design of captopril (1) was inspired by Byer’s independent report of D-benzylsuccinic acid (4) being a selective inhibitorof CPDA,163 where a carboxylate binds to Zn2þ and a benzyl side chain fits to the hydrophobic pocket of CPD (Scheme 17).157,158
Since the discovery of D-benzylsuccinic acid (4), all the inhibitors for CPDA comprise the 2-phenylpropanoic acid platform (5) (Scheme 17).164–173 Bartlettet al. have synthesized a tripep- tide analogue (5a) as a potent CPDA inhibitorhavingKiof 1 pM.164,165Kim’s group developed novel irreversible inhibitors such as 2-benzyl-3,4-epoxybutanoic acid (5c).166–170An X-ray crystal structure of (5c)-treated CPDA showed a covalent bond modification of the essential Glu270, which arose from the activation of the epoxide moiety by coordination to Zn2þof CPDA (Scheme 18a).164 Mobashery’s group synthesized 2-benzyl-3-iodopropanoic acid (5d) that inactivates CPDA irrevers- ibly.171The iodo moiety of (5d) could be activated by coordination to a Zn2þion (Scheme 18b).
A crystal structure of the (5d)-treated CPDA showed that Glu270 is alkylated by one enantiomer (corresponding to D-Phe) of (5d).172 The hydroxam derivative (5e) was also reported.173
Captopril (1)
IC50 = 23nM for ACE
R = Et: enalapril R = H: enalaprilat
(2a) (2b)
Trandolapril (3)
IC50 = 0.93nM for ACE IC50 = 4.5nM for ACE
Scheme 15
S1 S2 S2’
Substraste (angiotensin I)
Captopril (1)
Enalaprilat (2b) Inhibitors
ACE
Scheme 16