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 (P(1)) or position 22 (P'(1)) and used in steady-state kinetic analyses of ECE-1. The results indicate that the S(1) pocket of ECE-1 is relatively nonselective, but that the S'(1) 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 Arg(133) of ECE-1, which corresponds to Arg(102) 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 Arg(726) 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 (Arg(203)) and neprilysin (Arg(717)).
Johnson
Gary D
GD
Department of Biochemistry, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105, USA.
Swenson
Helen R
HR
Ramage
Robert
R
Ahn
Kyunghye
K
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D000072396:Endothelin-Converting Enzymes; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000235:genetics* / Q000378:metabolism*; D010449:Peptide Mapping
D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D015260:Neprilysin; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes