Molecular modelling and site-directed mutagenesis of the active site of endothelin-converting enzyme

Mammalian endothelin-converting enzyme is a membrane bound metalloprotease; its C-terminal domain contains sequence motifs characteristic of zinc meta lloproteases, We examined residues expected from molecular modelling to be important for substrate binding using selectively mutated recombinant rat E CE-1 alpha expressed in CHO cells. A conserved N-A-Ar-Ar (Ar = aromatic) mo tif is likely to be important for substrate binding. Mutating N550 to Gin o r Y552 to Phe reduces V-max/K-m by 8- and 18-fold, respectively, The equiva lent residue to Y553 in thermolysin binds the inhibitor through its NH grou p. Removing this putative interaction by mutating Tyr to Pro destroys activ ity, but mutating it to Ala or Phe also removes most activity. Mutating G58 3 (in a conserved GGI motif N-terminal of the zinc-binding helix) to Ala ha s no measurable effect, but mutating G584 to Ala destroys activity. Changin g V583 in the zinc-binding helix to Met, to mimic the sequence pattern in b ovine ECE-2, increases V-max/K-m to 1.7-fold that of the wild-type. Assays of phosphoramidon binding follow the pattern of those of substrate binding, but the IC50 of the more potent ECE inhibitor CGS 26303 was not significan tly altered by any of these mutations, suggesting that this compound may bi nd to ECE in a different mode from phosphoramidon.