Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, West Yorkshire, UK.
Mammalian endothelin-converting enzyme (ECE) is a membrane-bound zinc metalloprotease, and a member of the neprilysin sub-family. Its C-terminal domain has about 20% sequence identity to the bacterial metalloprotease, thermolysin, and contains sequence motifs characteristic of zinc metallo- proteases. From the sequence alignment of ECE with neprilysin and thermolysin, the structure of thermolysin and functional studies we constructed a preliminary molecular model of those residues likely to form the active site of ECE (Sansom et al. (1995), J. Cardiovasc. Pharmacol. 26S3, S75-S77). We examined those residues expected to be important for substrate and inhibitor binding using site directed mutagenesis with rat ECE-1.
A conserved N-A-Ar-Ar motif (residues 550-553# ; Ar = any aromatic) believed to form part of the S1 and S2 subsites is very important for activity. Changing N550 to Q or Y552 to F (the latter as in neprilysin or thermolysin) reduced specific activity to 17-20% of wild type. In thermolysin, the equivalent residue to Tyrosine 553 binds substrate or inhibitor through a water-mediated hydrogen bond from its backbone NH group. Removing this putative backbone interaction in rat ECE-1 by mutating Y553 to proline completely removes activity.
There is a conserved IGG motif N-terminal of the zinc-binding HExxH motif. Mutating G583 to Alanine has no measurable effect, but mutating G584 to Alanine removes almost all activity. Residue 589 is Valine in all ECE-1 sequences, but Methionine in bovine ECE-2. (This is the only change in any active site residue in ECE-2). Mutating this residue to Methionine in ECE-1 increases specific activity to about 350% of wild type. Tyrosine 633 is the most likely equivalent residue to Y157 in thermolysin, which can form hydrogen-bonds to a substrate or inhibitor. Mutating this residue to Phenylalanine reduces specific activity by about 20%. This change is compatible with the loss of a single hydrogen bond.
#: all numbering from rat ECE-1