Conformationally Regulated Peptide Bond Cleavage in Bradykinin

Abstract

Ion mobility and mass spectrometry techniques are used to investigate the stabilities of different conformations of bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). 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 Arg1-Pro2, resulting in the Arg1-cis-Pro2-cis-Pro3-Gly4-Phe5-Ser6-cis-Pro7-Phe8-Arg9 (all-cis) configuration. Once formed, the all-cis [BK+3H]3+ spontaneously cleaves the bond between Pro2-Pro3 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 Pro2-Pro3 occurs through multiple intermediates and is regulated by trans → cis isomerization of Arg1-Pro2. 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, ΔH‡ = 107.3 ± 9.2 kJ·mol–1, and ΔS‡ = 44.4 ± 5.1 J·mol–1·K–1 for bond cleavage. Biological resistance to the most favored intrinsic processing pathway prevents formation of Pro3-Gly4-Phe5-Ser6-cis-Pro7-Phe8-Arg9 that is approximately an order of magnitude more antigenic than BK.