Protein Engineering, Vol 11, 1235-1241, Copyright © 1998 by Oxford University Press
CE Sansom, MV Hoang and AJ Turner
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-1alpha 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 Vmax/Km 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 Vmax/Km 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.
ARTICLES
Molecular modelling and site-directed mutagenesis of the active site of endothelin-converting enzyme
School of Biochemistry and Molecular Biology, University of Leeds, UK.
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