Bradykinin analogues with β-amino acid substitutions reveal subtle differences in substrate specificity between the endopeptidases EC 3.4.24. 15 and EC 3.4.24.16

The closely related zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) cleave many common substrates, including bradykinin (BK). As such, there are few substrate-based inhibitors which are sufficiently selective to distinguish their activities. We have used BK analogues with either alanine or β-amino acid (containing an additional carbon within the peptide backbone) substitutions to elucidate subtle differences in substrate specificity between the enzymes. The cleavage of the analogues by recombinant EP24.15 and EP24.16 was assessed, as well as their ability to inhibit the two enzymes. Alanine-substituted analogues were generally better substrates than BK itself, although differences between the peptidases were observed. Similarly, substitution of the four N-terminal residues with β-glycine enhanced cleavage in some cases, but not others. β-Glycine substitution at or near the scissile bond (Phe⁵-Ser⁶) completely prevented cleavage by either enzyme; interestingly, these analogues still acted as inhibitors, although with very different affinities for the two enzymes. Also of interest, β-Gly⁸-BK was neither a substrate nor an inhibitor of EP24.15, yet could still interact with EP24.16. Finally, while both enzymes could be similarly inhibited by the D-stereoisomer of β-C3-Phe⁵-BK (IC₅₀ 20 μM, compared to 8 μM for BK), EP24.16 was relatively insensitive to the L-isomer (IC₅₀ ≃ 12 μM for EP24.15, > 40 μM for EP24.16). These studies indicate subtle differences in substrate specificity between EP24.15 and EP24.16, and suggest that β-amino acid analogues may be useful as templates for the design of selective inhibitors. Copyright (C) 2000 European Peptide Society and John Wiley and Sons, Ltd.