STRUCTURE-FUNCTION ANALYSIS OF ANGIOTENSIN I-CONVERTING ENZYME USING MONOCLONAL-ANTIBODIES - SELECTIVE-INHIBITION OF THE AMINO-TERMINAL ACTIVE-SITE

Angiotensin I-converting enzyme (ACE; kininase II) contains two very s imilar domains (the NH2- and COOH-terminal domains (N and C domains, r espectively)), each bearing an active site. These active sites hydroly ze the same peptides, but do not have the same catalytic properties an d substrate specificities. In an attempt to develop domain-specific im munological probes, two series of monoclonal antibodies (mAbs), 19 clo nes in all, were produced and tested against human ACE. These mAbs rec ognized at least nine different epitopes within three antigenic region s of the ACE molecule. Testing on wildtype recombinant ACE and several mutants with only one intact domain showed that these epitopes were a ll located in the N domain. None of the mAbs recognized the C domain. This particular specificity and analysis of results obtained with seve ral polyclonal antibodies to human ACE suggest that ACE immunogenicity is determined mainly by the N domain. Two mAbs (3A5 and i2H5) recogni zing epitopes from different antigenic regions of ACE inhibited the en zymatic activity of the N (but not of the C) domain. mAb 3A5 had the s ame inhibitory potency toward hippuryl-His-Leu, benzyloxycarbonyl-Phe- His-Leu, and angiotensin I hydrolysis, with 50% inhibition achieved at a mAb/ACE molar ratio of 6. mAb i2H5 was roughly three times more eff ective than mAb 3A5 in inhibiting the hydrolysis of benzyloxycarbonyl- Phe-His-Leu and the natural substrates angiotensin I and bradykinin (5 0% inhibition at a molar ratio of 1-2), but was less effective in inhi biting hippuryl-His-Leu cleavage (50% inhibition at a molar ratio of 2 2-25), indicating that this substrate interacts with a specific subsit e. mAb i2H5 almost completely inhibited the hydrolysis of the luteiniz ing hormone-releasing hormone by the isolated N domain. Both the prima ry carboxyl- and amino-terminal cleavages of this peptide were suppres sed. This antibody suppressed the primary amino-terminal cleavage of t he luteinizing hormone-releasing hormone by wild-type ACE by >90%, ind icating that this particular ACE function is mediated mainly by the N domain active site.These data provide evidence for structural differen ces between the two homologous domains of ACE despite their high degre e of sequence homology and show that monoclonal antibodies are able to distinguish between the two active sites in ACE.