DIFFERENCES IN THE PROPERTIES AND ENZYMATIC SPECIFICITIES OF THE 2 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 funct ional active site hydrolyzing Hip-His-Leu or angiotensin I (AI). The s ubstrate specificity of the two active sites of ACE was compared using wild-type recombinant ACE and mutants, where one active site is suppr essed by deletion or inactivated by mutations of 2 histidines coordina ting an essential zinc atom. Both active sites converted bradykinin (B K) to BK1-7 and BK1-5 with similar kinetics and with K(m)app at least 30 times lower and k(cat)/K(m)app 10 times higher than for AI. The car boxyl-terminal active site, but not the amino-terminal site, was activ ated by chloride; however, chloride activation was minimal compared wi th AI. Both domains also hydrolyzed substance P and cleaved a carboxyl -terminal protected dipeptide and tripeptide. The carboxyl-terminal ac tive site was more readily activated by chloride and hydrolyzed substa nce P faster. Luteinizing-hormone releasing hormone was hydrolyzed by both active sites, but hydrolysis by the amino-terminal active site wa s faster. It performed the endoproteolytic amino-terminal cleavage of this peptide at least 30 times faster than the carboxyl-terminal activ e 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. Howe ver, only the carboxyl-terminal active site can undergo a chloride-ind uced alteration that greatly enhances the hydrolysis of Al or substanc e P, and the amino-terminal active site possesses an unusual amino-ter minal 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.