Identification of an endothelin-converting enzyme-2-specific fluorigenic substrate and development of an in vitro and ex vivo enzymatic assay

J Biol Chem. 2010 Nov 5;285(45):34390-400. doi: 10.1074/jbc.M110.120576. Epub 2010 Aug 31.


Endothelin-converting enzyme-2 (ECE-2) is a membrane-bound zinc-dependent metalloprotease that shares a high degree of sequence homology with ECE-1, but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task that is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1-specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast® libraries using ECE-1 and ECE-2 allowed the identification of Ac-SKG-Pya-F-W-Nop-GGK-NH(2) (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (k(cat)/K(m)) of 8.1 ± 0.9 × 10(3) M(-1) s(-1). Using this novel substrate, we also characterized the first potent (K(i) = 7.7 ± 0.3 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knock-out tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.

MeSH terms

  • Animals
  • Aspartic Acid Endopeptidases / administration & dosage
  • Aspartic Acid Endopeptidases / biosynthesis*
  • Aspartic Acid Endopeptidases / chemistry*
  • Aspartic Acid Endopeptidases / genetics
  • Endothelin-Converting Enzymes
  • Endothelins / chemistry*
  • Endothelins / genetics
  • Endothelins / metabolism
  • Enzyme Assays / methods*
  • Enzyme Inhibitors / chemistry
  • Fluorescent Dyes / chemistry*
  • Gene Expression Regulation, Enzymologic / physiology*
  • Humans
  • Metalloendopeptidases / administration & dosage
  • Metalloendopeptidases / biosynthesis*
  • Metalloendopeptidases / chemistry*
  • Metalloendopeptidases / genetics
  • Mice
  • Organ Specificity
  • Peptide Library
  • Peptides / chemistry*
  • Peptides / genetics
  • Peptides / metabolism
  • Substrate Specificity


  • Endothelins
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Peptide Library
  • Peptides
  • Aspartic Acid Endopeptidases
  • Metalloendopeptidases
  • ECE1 protein, human
  • ECE2 protein, human
  • Ece1 protein, mouse
  • Ece2 protein, mouse
  • Endothelin-Converting Enzymes