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
Ra. Lew et al., 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, J PEPT SCI, 6(9), 2000, pp. 440-445
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.