Conformationally Regulated Peptide Bond Cleavage in Bradykinin

摘要

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), 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 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.