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, 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.

Substance P in Solution: Trans-to-Cis Configurational Changes of Penultimate Prolines Initiate Non-enzymatic Peptide Bond Cleavages

“…[20] This mechanism requires a cisconfigured peptide bond between the first and second residues (e.g., Xxx 1 -cis-Xxx 2 , where Xxx is any amino acid) -the same configuration that we assigned to a critical intermediate for cleavage of the peptide bond of bradykinin. [10] Capasso also found that cyclization of an Ala 1 -Pro 2 -NH 2 dipeptide has a significant pH dependence under more basic conditions, but was pH-independent in acidic conditions. [20] These results appear to be sequence dependent, as analysis of His 1 -Pro 2 -NH 2 cyclization produces a more bell-shaped pH dependence profile.…”

“…Below, we show that subP undergoes sequential bond cleavages, producing both cyclo-Arg 1 -Pro 2 (cRP) and cyclo-Lys 3 -Pro 4 (cKP) in the form of DKPs. Similar to bradykinin, [10] the first cleavage is preceded by a configurationally-coupled protonation event. While the second cleavage involves only one charge state, a long induction period prior to cleavage is observed.…”

“…Recently, this approach was used to study the degradation of the nonapeptide bradykinin. [10] When heated, in the absence of enzymes, bradykinin undergoes a spontaneous and highly-specific bond cleavage to produce the cyclo-Arg 1 -Pro 2 DKP product. Upon monitoring the abundances of species that are resolved in the IMS-MS spectrum over time, we found evidence for at least five resolvable steps for this reaction, including a slow configurationally-coupled protonation.…”

“…Spontaneous formation of DKP is not limited to subP and bradykinin. [10] Recombinant human growth hormone undergoes a similar reaction in the absence of enzymes. [12] DKP is observed as a side product in laboratory peptide synthesis and necessitates that certain precautions are taken to minimize its formation.…”

Allosteric inhibition of the SARS-CoV-2 main protease – insights from mass spectrometry-based assays

“…Quenching the reaction enables interrogation of these transient species captured in the MS instrument. [9] A mass spectrum collected 30 s after initiating the cleavage reaction is shown (Figure 3). Several satellite peaks are observed alongside the main charge state series, one of which corresponds to the enzyme substrate complex with a mass of 68,781 ± 2 Da, in agreement with dimeric M Pro bound to a single 11-mer substrate (1192 Da).…”