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.

Angiotensin I-converting enzyme (ACE, E.C.3.4.15.1) has been recently shown to contain two very similar domains, each of which bears a functional active site hydrolyzing Hip-His-Leu or angiotensin I (AI). The substrate specificity of the two active sites of ACE was compared using wild-type recombinant ACE and mutants, where one active site is suppressed by deletion or inactivated by mutations of 2 histidines coordinating an essential zinc atom. Both active sites converted bradykinin (BK) to BK1-7 and BK1-5 with similar kinetics and with Kappm at least 30 times lower and kcat/kappm 10 times higher than for AI. The carboxyl-terminal active site, but not the amino-terminal site, was activated by chloride; however, chloride activation was minimal compared with AI. Both domains also hydrolyzed substance P and cleaved a carboxyl-terminal protected dipeptide and tripeptide. The carboxyl-terminal active site was more readily activated by chloride and hydrolyzed substance P faster. Luteinizing-hormone releasing hormone was hydrolyzed by both active sites, but hydrolysis by the amino-terminal active site was faster. It performed the endoproteolytic amino-terminal cleavage of this peptide at least 30 times faster than the carboxyl-terminal active site. Both active sites cleaved a carboxyl-terminal tripeptide from luteinizing hormone-releasing hormone. Thus, both active sites of ACE possess dipeptidyl carboxypeptidase and endopeptidase activities. However, only the carboxyl-terminal active site can undergo a chloride-induced alteration that greatly enhances the hydrolysis of AI or substance P, and the amino-terminal active site possesses an unusual amino-terminal endoproteolytic specificity for a natural peptide. This suggests physiologically important differences between the subsites of the two active centers, and different substrate specificity, despite the high degree of sequence homology.