The hydrolysis of endothelins by neutral endopeptidase 24.11 (Enkephalinase)

Endothelins 1-3 are a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear 'COOH-terminal tail'. These peptides were originally described on the basis of their potent vasoconstrictor activity. The hydrolytic inactivation of endothelin action has recently been implicated to be attributed, at least in part, to the enzyme neutral endopeptidase 24.11 (Scicli, A.G., Vijayaraghavan, Hersh, L., and Carretero, O. (1989) Hypertension 14, 353). The kinetic properties and mode of hydrolysis of the endothelins by this enzyme are reported in this study. The K(m) for endothelins 1 and 3 hydrolysis is approximately 2 μM while endothelin² exhibits a 5-fold higher K(m). Endothelins 1 and 2 exhibit similar V(max) values while endothelin³ is hydrolyzed considerably more slowly. The initial cleavage site in endothelin¹ is at the Ser⁵-Leu⁶ bond located within one of the cyclic structures. Thermolysin, a bacterial neutral endopeptidase with a similar substrate specificity to neutral endopeptidase 24.11 initially cleaves endothelin¹ between His¹⁶-Leu¹⁷ which lies within the COOH-terminal linear 'tail' portion of the molecule. The cleavage of endothelins 2 and 3 by neutral endopeptidase 24.11 differs from that observed with endothelin¹ in that cleavage of these endothelins occurs at Asp¹⁸-Ile¹⁹ within the linear COOH-terminal tail structure. These results demonstrate that the endothelins are good substrates for neutral endopeptidase 24.11 and suggest that their mode of cleavage is dependent upon both amino acid sequence as well as peptide conformation.