Mapping the Active Site of Endothelin-Converting Enzyme-1 through Subsite Specificity and Mutagenesis Studies: A Comparison with Neprilysin

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound zinc metallopeptidase that is homologous to neprilysin in amino acid sequence. A major in vivo function of ECE-1 is the generation of endothelin-1, a potent vasoconstrictor, from big endothelin-1. ECE-1 is also potentially involved in the processing or degradation of other peptide hormones. In this study we have used substrates based on the sequence of the COOH-terminal half of big endothelin-1 to examine the subsite specificity of recombinant ECE-1. The big endothelin-1 [16–38] peptides were systematically varied at either position 21 (P1) or position 22 (P1′) and used in steady-state kinetic analyses of ECE-1. The results indicate that the S1 pocket of ECE-1 is relatively nonselective, but that the S1′ subsite of ECE-1 has a preference for large hydrophobic side chains. The peptidyl carboxydipeptidase activity of ECE-1 was also characterized, revealing that substrates with COOH-terminal carboxylates are highly preferred over the cognate amides and esters. A site-directed mutagenesis study was carried out to identify the active-site amino acid residues specifically involved in binding to the COOH-terminal carboxylate of substrates. The data indicate that Arg133 of ECE-1, which corresponds to Arg102 of neprilysin that has been identified as an active-site residue of neprilysin involved in binding to the free carboxylate of some substrate peptides, may not play the same role. However, the low activity observed for an ECE-1 Arg726 mutant is consistent with a role for this arginine residue in the binding of substrates, a role which has been ascribed to arginine residues in both thermolysin (Arg203) and neprilysin (Arg717).