Bradykinin analogues with beta-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 E C 3.4.24.16 [EP24.16) cleave many common substrates, including bradykinin ( BK). As such, there are few substrate-based inhibitors which are sufficient ly selective to distinguish their activities. We have used BK analogues wit h either alanine or beta-amino acid [containing an additional carbon within the peptide backbone) substitutions to elucidate subtle differences in sub strate specificity between the enzymes. The cleavage of the analogues by re combinant EP24.15 and EP24.16 was assessed, as well as their ability to inh ibit the two enzymes. Alanine-substituted analogues were generally better s ubstrates than BK itself, although differences between the peptidases were observed. Similarly, substitution of the four N-terminal residues with beta -glycine enhanced cleavage in some cases, but not others. beta-Glycine subs titution at or near the scissile bond (Phe(5)-Ser(6)) completely prevented cleavage by either enzyme: interestingly, these analogues still acted as in hibitors, although with very different affinities for the two enzymes. Also of interest, beta-Gly(8)-BK was neither a substrate nor an inhibitor of EP 24.15, yet could still interact with EP24.16. Finally, while both enzymes c ould be similarly inhibited by the D-stereoisomer of beta-C3-Phe(5)-BK [IC5 0 approximate to 20 mu M, compared to 8 mu M for BK), EP24.16 was relativel y insensitive to the L-isomer (IC50 approximate to 12 mu M for EP24.15. > 4 0 mu M for EP24.16). These studies indicate subtle differences in substrate specificity between EP24.15 and EP24.16, and suggest that beta-amino acid analogues may be useful as templates for the design of selective inhibitors . Copyright (C) 2000 European Peptide Society and John Wiley & Sons, Ltd.