Endothelin-Converting Enzyme: Substrate Specificity and Inhibition by Novel Analogs of Phosphoramidon

Abstract Endothelin converting enzyme was partially purified by detergent extraction and ion exchange chromatography from porcine aortic endothelial cells. This kinetically homogeneous preparation catalyzes the hydrolysis of porcine big endothelin-1 to endothelin-1 with a pH optimum of 7. Human big endothelins-1, -2, and -3 are also hydrolyzed, but at progressively lower rates. Fragments of big porcine endothelin-1 comprising residues 16–39 and 16–29 are good substrates, but additional C -terminal truncations are devoid of substrate activity. Endothelin converting enzyme is characteristically inhibited by phosphoramidon and other metalloproteinase inhibitors including EDTA, o -phenanthroline, and diethylpyrocarbonate, but not by inhibitors of other classes of proteases or thiorphan. The inhibition by phosphoramidon is competitive with big porcine endothelin-1 suggestive of a common binding site for substrate and inhibitor. A number of novel analogs of phosphoramidon were synthesized by modifying various regions of the molecule and tested for inhibitory activity. The most potent of these, a methylphosphonic acid, has an IC 50 of 0.05 μM.