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

We report ion mobility spectrometry and mass spectrometry studies of the non-enzymatic step-by-step degradation of substance P (subP), an 11-residue neuropeptide, with the sequence Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH, in ethanol. At elevated solution temperatures (55 to 75 °C), several reactions are observed, including a protonation event, i.e., [subP+2H] + H → [subP+3H], that appears to be regulated by a configurational change and two sequential bond cleavages (the Pro-Lys peptide bond is cleaved to form the smaller nonapeptide Lys-Met-NH [subP], and subsequently, subP is cleaved at the Pro-Gln peptide bond to yield the heptapeptide Gln-Met-NH [subP]). Each of the product peptides [subP and subP] is accompanied by a complementary diketopiperazine (DKP): cyclo-Arg-Pro (cRP) for the first cleavage, and cyclo-Lys-Pro (cKP) for the second. Insight about the mechanism of degradation is obtained by comparing kinetics calculations of trial model mechanisms with experimental data. The best model of our experimental data indicates that the initial cleavage of subP is regulated by a conformational change, likely a trans→cis isomerization of the Arg-Pro peptide bond. The subP product has a long lifetime (t ~ 30 h at 55 °C) and appears to transition through several structural intermediates prior to dissociation, suggesting that subP is initially formed with a Lys-trans-Pro peptide bond configuration and that slow trans→cis isomerization regulates the second bond cleavage event as well. From these data and our model mechanisms, we obtain transition state thermochemistry ranging from ΔH = 41 to 85 kJ mol and ΔS = - 43 to - 157 J mol K for each step in the reaction. Graphical Abstract.