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-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). At elevated solution temperatures, we observe a slow protonation reaction, i.e., [BK+2H]+H → [BK+3H], that is regulated by trans → cis isomerization of Arg-Pro, resulting in the Arg- cis-Pro- cis-Pro-Gly-Phe-Ser- cis-Pro-Phe-Arg (all- cis) configuration. Once formed, the all- cis [BK+3H] spontaneously cleaves the bond between Pro-Pro 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-Pro occurs through multiple intermediates and is regulated by trans → cis isomerization of Arg-Pro. From this mechanism, we can extract transition state thermochemistry: Δ G = 94.8 ± 0.2 kJ·mol, Δ H = 79.8 ± 0.2 kJ·mol, and Δ S = -50.4 ± 1.7 J·mol·K for the trans → cis protonation event; and, Δ G = 94.1 ± 9.2 kJ·mol, Δ H = 107.3 ± 9.2 kJ·mol, and Δ S = 44.4 ± 5.1 J·mol·K for bond cleavage. Biological resistance to the most favored intrinsic processing pathway prevents formation of Pro-Gly-Phe-Ser- cis-Pro-Phe-Arg that is approximately an order of magnitude more antigenic than BK.