The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity

Abstract

sACE (somatic angiotensin-converting enzyme) consists of two homologous, N and C domains, whereas the testis isoenzyme [tACE (testis ACE)] consists of a single C domain. Both isoenzymes are shed from the cell surface by a sheddase activity, although sACE is shed much less efficiently than tACE. We hypothesize that the N domain of sACE plays a regulatory role, by occluding a recognition motif on the C domain required for ectodomain shedding and by influencing the catalytic efficiency. To test this, we constructed two mutants: CNdom-ACE and CCdom-ACE. CNdom-ACE was shed less efficiently than sACE, whereas CCdom-ACE was shed as efficiently as tACE. Notably, cleavage occurred both within the stalk and the interdomain bridge in both mutants, suggesting that a sheddase recognition motif resides within the C domain and is capable of directly cleaving at both positions. Analysis of the catalytic properties of the mutants and comparison with sACE and tACE revealed that the k(cat) for sACE and CNdom-ACE was less than or equal to the sum of the kcat values for tACE and the N-domain, suggesting negative co-operativity, whereas the kcat value for the CCdom-ACE suggested positive co-operativity between the two domains. Taken together, the results provide support for (i) the existence of a sheddase recognition motif in the C domain and (ii) molecular flexibility of the N and C domains in sACE, resulting in occlusion of the C-domain recognition motif by the N domain as well as close contact of the two domains during hydrolysis of peptide substrates.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…In contrast, the N-domain may lack such recognition sequences. Consistent with this is the finding that an ACE construct, composed of a tandem repeat of two ACE C-domains, is shed from cells with kinetics similar to the testis ACE protein, which contains only a single C-domain (Woodman et al, 2005). Not only was the repeated Cdomain construct shed, the sheddase enzyme cut both within the ACE stalk region and in the interdomain 8 protein bridge connecting the two C-domains.…”

“…Limited proteolysis of ACE with endoproteinase Asp-N will cleave between the Thr615-Asp616 and the Leu1219-Asp1220 peptide bonds to generate the two catalytic domains in active form that can be separated by a lisinopril affinity column Chen et al, 2010). Several groups have suggested that in somatic ACE, the catalytic activity of the C-domain is negatively regulated by the presence of the N-domain (Binevski et al, 2003;Woodman et al, 2005).…”

Porcine pulmonary angiotensin I-converting enzyme—Biochemical characterization and spatial arrangement of the N- and C-domains by three-dimensional electron microscopic reconstruction

“…2) which was eluted with 20mM Mes-NaOH (pH 6.0) and 10 µM ZnCl2 represents the 90 kDa N-domain ( fig.3; (Table 5) indicated that the catalytic efficiency (Kcat/Km) of the isolated Cdomain is about two-fold higher than that of the native sACE. These results are compatible with the notion that the catalytic activity of the C-domain is negatively regulated by it's Ndomain counterpart in native sACE (Binevski et al, 2003;Woodman et al, 2005;Skirgello et al, 2005).…”

“…Although human as well as bovine sACE display negative cooperativity between the N-and Cdomains' active sites upon binding of diverse peptides or inhibitors (Binevski et al, 2003;Woodman et al, 2005;Skirgello et al, 2005), the enzymic activities are not linked to the glycosylation state. Deglycosylation of sACE from porcine lung did not markedly affect the biochemistry or structural properties of the enzyme (Baudin et al, 1997).…”

Shedding the load of hypertension: The proteolytic processing of angiotensin-converting enzyme

“…Work from our and other laboratories has indicated that this is most likely because there is a sheddase recognition domain in the C domain of ACE, which in somatic ACE is partly occluded by the presence of the second domain (N domain). 8,9 Indeed, when a mutant ACE was constructed in which the N domain of somatic ACE was fused directly to the stalk, this mutant was not shed, suggesting that it lacked a critical recognition element for the sheddase. …”