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: Δ G^‡ = 94.8 ± 0.2 kJ·mol^-1, Δ H^‡ = 79.8 ± 0.2 kJ·mol^-1, and Δ S^‡ = -50.4 ± 1.7 J·mol^-1·K^-1 for the trans → cis protonation event; and, Δ G^‡ = 94.1 ± 9.2 kJ·mol^-1, Δ H^‡ = 107.3 ± 9.2 kJ·mol^-1, and Δ 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 Pro³-Gly⁴-Phe⁵-Ser⁶- cis-Pro⁷-Phe⁸-Arg⁹ that is approximately an order of magnitude more antigenic than BK.