Current Topics in Medicinal Chemistry 2004, 4, 403-429 403 Structural Features of Angiotensin-I Converting Enzyme Catalytic Sites: Conformational Studies in Solution, Homology Models and Comparison with Other Zinc Metallopeptidases Georgios A. Spyroulias*,1, Athanassios S. Galanis1, George Pairas1, Evy Manessi-Zoupa2 and Paul Cordopatis*,1 Departments of Pharmacy1 and Chemistry2, University of Patras, GR-26504, Patras, GREECE Abstract: Angiotensin-I Converting Enzyme (ACE) is a Zinc Metallopeptidase of which the three-dimensional stucture was unknown until recently, when the Xray structure of testis isoform (C-terminal domain of somatic) was determined. ACE plays an important role in the regulation of blood pressure due to its action in the frame of the Renin-Angiotensin System. Efforts for the specific inhibition of the catalytic function of this enzyme have been made on the basis of the X- ray structures of other enzymes with analogous efficacy in the hydrolytic cleavage of peptide substrate terminal fragments. Angiotensin-I Converting Enzyme bears the sequence and topology characteristics of the well-known gluzincins, a sub-family of zincins metallopeptidases and these similarities are exploited in order to reveal common structural elements among these enzymes. 3D homology models are also built using the X-ray structure of Thermolysin as template and peptide models that represent the amino acid sequence of the ACE’s two catalytic, zinc-containing sites are designed and synthesized. Conformational analysis of the zinc-free and zinc-bound peptides through high resolution 1H NMR Spectroscopy provides new insights into the solution structure of ACE catalytic centers. Structural properties of these peptides could provide valuable information towards the design and preparation of new potent ACE inhibitors. 1. INTRODUCTION the last 30 years has been achieved through inhibitors based on the pioneering work of Ferreira S.H. [15] and Ondetti 1.1. Angiotensin-I Converting Enzyme and Hypertension M.A. [16,17]. These researchers showed that the venom of a Angiotensin-I Converting Enzyme (ACE), isolated in the Brazilian pit viper contained a factor that greatly enhanced mid 50’s, is a Zinc Metallopeptidase and one of the major the smooth-muscle-relaxing action of the nonapeptide BK components of the so-called Renin-Angiotensin System which also inhibiting ACE. All ACE inhibitors were (RAS) [1-3]. Renin is responsible for the liberation of prepared in the absence of the ACE’s three-dimensional Angiotensin I (AI) in blood, after renin’s catalytic action on structure and bear two main characteristics: (i) designed the angiotensinogen. ACE possesses a crucial role in the on the basis of venom peptide extracting structural regulation of blood pressure since it catalyzes the cleavage of information for the enzyme catalytic site from the crystal the C-terminal His-Leu dipeptide of the rather inactive structure of Carboxypeptidase A (CPA) [18], and (ii) high decapeptide Angiotensin I (AI), in the vasopressor biological activity strongly coupled with enhanced zinc octapeptide Angiotensin II (AII) [4-6] (Fig. (1)). However, binding ability [19]. ACE impact is not only focused on the generation of Angiotensin II but also extended to inactivation of the 1.2. ACE Inhibitors and their Impact in Medicine and vasodilator peptides Bradykinin (BK) and Kallidin [7-9]. Pharmacology ACE is encountered in two distinct forms in humans, the ACE inhibitors are considered among the most potent somatic and the testis form. These differ from the structural antihypertensive drugs and apart their major action, exhibit point of view, mainly in size and number of catalytic sites beneficial lateral effects in the prevention of cardiovascular [10,11]. According to its function, ACE is classified among disease in various classes of hypertensive patients. the peptidyl dipeptidases of zinc metallopeptidases super- Additionally ACE inhibitors have been proven more family due to its ability to remove C-terminal dipeptide [5] effective than other hypertensive substances in reducing from substrates. ACE can also exhibit activity of an proteinuria and retarding the progression of renal damage in endopeptidase against substrates such as Substance P, patients with various types of nephropathy. These features Cholecystokinin and Luliberin (LHRH), peptides with are probably among the reasons that two (International amidated C-end [9,12,13]. As far as the ACE role in blood Society of Hypertension-World Health Organization, ISH- pressure is concerned, the inhibition of ACE enzymatic WHO; Canadian Society of Hypertension) of the three health activity against AI was considered as one of the major organisations (the third is the British Hypertension Society) challenges against hypertensive disease and congestive heart affiliated with hypertension, recommend ACE inhibitors in failure [14]. Therapy, today and after extensive research for the first-line of antihypertensive drug treatment, after the results of the first trials of this type of inhibitors became *Address correspondence to these authors at Department of Pharmacy, available at 1996. University of Patras, GR-265 04, Greece; Tel: +30 2610 997 721; Fax: +30 An alternative treatment of hypertension is focused on 2610 997 714; e-mail: [email protected] and [email protected] the blockade of AII receptors, AT1R and AT2R (94% amino 1568-0266/04 $45.00+.00 © 2004 Bentham Science Publishers Ltd. 404 Current Topics in Medicinal Chemistry, 2004, Vol. 4, No. 4 Spyroulias et al. Fig. (1). Schematic representation of the Renin-Angiotensin system and the role of the two enzymes, Renin and Angiotensin Converting Enzyme (ACE) on regulation of blood pressure through the generation and release of Angiotensin II vassopressor peptide. acid sequence identity), with appropriate antagonists. The the “spacers”. Zinc sites are also characterised by secondary class of AT1R which are currently under continuous interactions with neighbouring amino acids that position in development and trials, exhibit at the moment only one main space and conformational features strongly depend on the the advantage over ACE inhibitors, which is the absence of overall protein folding and three-dimensional structure. cough as a side effect. Interestingly, only ISH-WHO has These features are critical for the structure-function relationship of this class of metalloenzymes and dictate their recommended AT1R antagonists as first line antihypertensive drugs, and this is probably due to the absence of any classification into various families. published long-term trial results. For all these reasons, the The characteristic amino acid sequences, which contain design and preparation of new potent ACE inhibitors still the potential three zinc ligands in the zinc metallopeptidase remains one of the main challenges in the intersection of the family, comprise the binding motif sequences that are a fields of chemistry, pharmacology and medicine. diagnostic tool in enzyme classification. The first two of the protein ligands are found in the first three-, four- or five- 1.3. Zinc Catalytic Sites and their Characteristics residue binding motif, while the third is found in a second characteristic motif. These residue-ligands are generally Zinc sites in metalloenzymes and related biomolecules separated either by short amino acid “spacers” among the are classified according to their ligands and coordination first, second and third ligands, or by a short spacer between geometry into three types of zinc binding sites [20,21]: (i) the two first ligands and by a large spacer between the the (ii) the and (iii) the catalytic, cocatalytic structural. second and third, or fourth ligand, should one exist. The Hydrolases like ACE possess a zinc site which catalytic magnitude of a short spacer could vary from one to three usually coordinates with nitrogen, oxygen and sulphur amino acids between the first two ligands. On the other hand, donors of His, Glu, Asp and Cys residues while His is most the long spacers usually found in various metallopeptidase frequently encountered in the coordination sphere of zinc subfamilies could vary from 5 to over 100 amino acids. The metal ion. Water is also a zinc ligand in catalytic sites and is length of the spacer between the two first ligands belonging activated for ionisation, polarisation, or displacement by the to the same binding motif often characterizes the secondary identity and arrangement of ligands coordinated with zinc structure of this protein fragment. For example, a three- [22]. The zinc coordination number for this kind of sites residue spacer is characteristic of a a -helix conformation has been found to be four or five and the donor atoms of while a one-residue spacer indicates a b sheet conformation. residues define a distorted-tetrahedral or trigonal-bipyramidal coordination geometry. Ionisation and/or polarisation of the 1.4. Focus of this Article activated H2O is assisted by the base form of an active site- residue or in some cases by a “second-shell” residue This article aims to provide new structural insights into that yields hydroxide ions at neutral pH while water ACE, an enzyme whose role in hypertension has stimulated displacement results in Lewis acid catalysis on the part of the over the last 35 years extensive and continuous effort catalytic zinc metal. towards designing its potential inhibitors, even without the most important tool in the hands of biochemists, The structure of the zinc catalytic sites comprises: (i) the enzymologists