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

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 Arg¹-Pro²-Lys³-Pro⁴-Gln⁵-Gln⁶-Phe⁷-Phe⁸-Gly⁹-Leu¹⁰-Met¹¹-NH₂, in ethanol. At elevated solution temperatures (55 to 75 °C), several reactions are observed, including a protonation event, i.e., [subP+2H]²⁺ + H⁺ → [subP+3H]³⁺, that appears to be regulated by a configurational change and two sequential bond cleavages (the Pro²-Lys³ peptide bond is cleaved to form the smaller nonapeptide Lys³-Met¹¹-NH₂ [subP_(3-11)], and subsequently, subP_(3-11) is cleaved at the Pro⁴-Gln⁵ peptide bond to yield the heptapeptide Gln⁵-Met¹¹-NH₂ [subP_(5-11)]). Each of the product peptides [subP_(3-11) and subP_(5-11)] is accompanied by a complementary diketopiperazine (DKP): cyclo-Arg¹-Pro² (cRP) for the first cleavage, and cyclo-Lys³-Pro⁴ (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 Arg¹-Pro² peptide bond. The subP_(3-11) product has a long lifetime (t_1/2 ~ 30 h at 55 °C) and appears to transition through several structural intermediates prior to dissociation, suggesting that subP_(3-11) is initially formed with a Lys³-trans-Pro⁴ 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^-1 and ΔS^‡ = - 43 to - 157 J mol^-1 K^-1 for each step in the reaction. Graphical Abstract.