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

Abstract

Mammalian endothelin-converting enzyme is a membrane-bound metalloprotease; its C-terminal domain contains sequence motifs characteristic of zinc metalloproteases. We examined residues expected from molecular modelling to be important for substrate binding using selectively mutated recombinant rat ECE-1α expressed in CHO cells. A conserved N-A-Ar-Ar (Ar = aromatic) motif is likely to be important for substrate binding. Mutating N550 to Gln or Y552 to Phe reduces V(max)/K(m) by 8- and 18-fold, respectively. The equivalent residue to Y553 in thermolysin binds the inhibitor through its NH group. Removing this putative interaction by mutating Tyr to Pro destroys activity, but mutating it to Ala or Phe also removes most activity. Mutating G583 (in a conserved GGI motif N-terminal of the zinc-binding helix) to Ala has no measurable effect, but mutating G584 to Ala destroys activity. Changing V583 in the zinc-binding helix to Met, to mimic the sequence pattern in bovine 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 significantly altered by any of these mutations, suggesting that this compound may bind to ECE in a different mode from phosphoramidon.