Angiotensin-converting enzyme 2 (ACE2), a homologue of ACE, represents a new and potentially important target in cardio-renal disease. A model of the active site of ACE2, based on the crystal structure of testicular ACE, has been developed and indicates that the catalytic mechanism of ACE2 resembles that of ACE. Structural differences exist between the active site of ACE (dipeptidyl carboxypeptidase) and ACE2 (carboxypeptidase) that are responsible for the differences in specificity. The main differences occur in the ligand-binding pockets, particularly at the S2' subsite and in the binding of the peptide carboxy-terminus. The model explains why the classical ACE inhibitor lisinopril is unable to bind to ACE2. On the basis of the ability of ACE2 to cleave a variety of biologically active peptides, a consensus sequence of Pro-X-Pro-hydrophobic/basic for the protease specificity of ACE2 has been defined that is supported by the ACE2 model. The dipeptide, Pro-Phe, completely inhibits ACE2 activity at 180 microM with angiotensin II as the substrate. As with ACE, the chloride dependence of ACE2 is substrate-specific such that the hydrolysis of angiotensin I and the synthetic peptide substrate, Mca-APK(Dnp), are activated in the presence of chloride ions, whereas the cleavage of angiotensin II is inhibited. The ACE2 model is also suggestive of a possible mechanism for chloride activation. The structural insights provided by these analyses for the differences in inhibition pattern and substrate specificity among ACE and its homologue ACE2 and for the chloride dependence of ACE/ACE2 activity are valuable in understanding the function and regulation of ACE2.
A human zinc metalloprotease (termed ACEH or ACE2) with considerable homology to angiotensin-converting enzyme (ACE) (EC 3.4.15.1) has been identified and subsequently cloned and functionally expressed. The translated protein contains an N-terminal signal sequence, a single catalytic domain with zinc-binding motif (HEMGH), a transmembrane region, and a small C-terminal cytosolic domain. Unlike somatic ACE, ACEH functions as a carboxypeptidase when acting on angiotensin I and angiotensin II or other peptide substrates. ACEH may function in conjunction with ACE and neprilysin in novel pathways of angiotensin metabolism of physiological significance. In contrast with ACE, ACEH does not hydrolyse bradykinin and is not inhibited by typical ACE inhibitors. ACEH is unique among mammalian carboxypeptidases in containing an HEXXH zinc motif but, in this respect, resembles a bacterial enzyme, Thermus aquaticus (Taq) carboxypeptidase (EC 3.4.17.19). Collectrin, a developmentally regulated renal protein, is homologous with the C-terminal region of ACEH but has no similarity with ACE and no catalytic domain. Thus, the ACEH protein may have evolved as a chimera of a single ACE-like domain and a collectrin domain. The collectrin domain may regulate tissue response to injury whereas the catalytic domain is involved in peptide processing events.
Angiotensin-converting enzyme-2 (ACE2) is a membrane protein with its active site exposed to the extracellular surface of endothelial cells, the renal tubular epithelium and also the epithelia of the lung and the small intestine [1][2][3]. Here ACE2 is poised to metabolize circulating peptides which may include angiotensin II, a potent vasoconstrictor and the product of angiotensin I cleavage by angiotensin-converting enzyme (ACE; EC 3.4.15.1) [1,4]. Indeed, ACE2 has been implicated in the regulation of heart and renal function where it is proposed to control the concentrations of angiotensin II relative to its hypotensive metabolite, angiotensin-(1-7) [5][6][7][8][9][10][11][12][13]. Most recently, ACE2 has been identified as a functional receptor for the coronavirus which causes the severe acute respiratory syndrome (SARS) [14]. For recent reviews, see [15,16].ACE2 shares a number of characteristics with ACE, both being zinc-containing enzymes which are sensitive to anion activation [4,17,18]. However, unlike ACE, ACE2 functions as a carboxypeptidase and is not susceptible to inhibition by the classical ACE inhibitors [1,2]. After the elucidation of the crystal structure of testicular ACE (tACE), [19] a model of the active site of ACE2 was described which demonstrated the structural determinants underlying these differences in enzyme activity [17]. Critical residue substitutions were highlighted that gave rise to the elimination of the S2¢ pocket found in ACE such that ACE2 is able to remove only a single amino acid from the C-terminus of its substrates (whereas ACE is a peptidyl dipeptidase). Shortly after this, the structure of ACE2 was solved [20] which provided further insights into this enzyme in relation to its counterpart. However, it has Angiotensin-converting enzyme-2 (ACE2) may play an important role in cardiorenal disease and it has also been implicated as a cellular receptor for the severe acute respiratory syndrome (SARS) virus. The ACE2 activesite model and its crystal structure, which was solved recently, highlighted key differences between ACE2 and its counterpart angiotensin-converting enzyme (ACE), which are responsible for their differing substrate and inhibitor sensitivities. In this study the role of ACE2 active-site residues was explored by site-directed mutagenesis. Arg273 was found to be critical for substrate binding such that its replacement causes enzyme activity to be abolished. Although both His505 and His345 are involved in catalysis, it is His345 and not His505 that acts as the hydrogen bond donor ⁄ acceptor in the formation of the tetrahedral peptide intermediate. The difference in chloride sensitivity between ACE2 and ACE was investigated, and the absence of a second chloride-binding site (CL2) in ACE2 confirmed. Thus ACE2 has only one chloride-binding site (CL1) whereas ACE has two sites. This is the first study to address the differences that exist between ACE2 and ACE at the molecular level. The results can be applied to future studies aimed at unravelling the role of ACE2, ...
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