Conformationally Regulated Peptide Bond Cleavage in Bradykinin.
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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: G = 94.8 0.2 kJmol-1, H = 79.8 0.2 kJmol-1, and S = -50.4 1.7 Jmol-1K-1 for the trans cis protonation event; and, G = 94.1 9.2 kJmol-1, H = 107.3 9.2 kJmol-1, and S = 44.4 5.1 Jmol-1K-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.
