Conformationally Regulated Peptide Bond Cleavage in Bradykinin

Ion mobility and mass spectrometry techniques are used to investigate the stabilities of different conformations of bradykinin (BK, Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)). At elevated solution temperatures, we observe a slow protonation reaction, i.e., [BK+2H](2+)+H(+) → [BK+3H](3+), that is regulated by trans → cis isomerization of Arg(1)-Pro(2), resulting in the Arg(1)-Cis-Pro(2)-cis-Pro(3)-Gly(4)-Phe(5)-Ser(6)-cis-Pro(7)-Phe(8)-Arg(9) (all-cis) configuration. Once formed, the all-cis [BK+3H](3+) spontaneously cleaves the bond between Pro(2)-Pro(3) with perfect specificity, a bond that is biologically resistant to cleavage by any human enzyme. Temperature-dependent kinetics studies reveal details about the intrinsic peptide processing mechanism. We propose that nonenzymatic cleavage at Pro(2)-Pro(3) occurs through multiple intermediates and is regulated by trans → cis isomerization of Arg(1)-Pro(2). From this mechanism, we can extract transition state thermochemistry: ΔG(‡) = 94.8 ± 0.2 kJ·mol(−1), ΔH(‡) = 79.8 ± 0.2 kJ·mol(−1), and ΔS(‡) = −50.4 ± 1.7 J·mol(−1)·K(−1) for the trans → cis protonation event; and, ΔG(‡) = 94.1 ± 9.2 kJ·mol(−1), →H(‡) = 107.3 ± 9.2 kJ·mol(−1), and →S(‡) = 44.4 ± 5.1 J·mol(−1)·K(−1) for bond cleavage. Biological resistance to the most favored intrinsic processing pathway prevents formation of Pro(3)-Gly(4)-Phe(5)-Ser(6)-cis-Pro(7)-Phe(8)-Arg(9) that is approximately an order of magnitude more antigenic than BK.