Phosphate-Catalyzed Succinimide Formation from Asp Residues: A Computational Study of the Mechanism

Abstract

Aspartic acid (Asp) residues in proteins and peptides are prone to the non-enzymatic reactions that give biologically uncommon l-β-Asp, d-Asp, and d-β-Asp residues via the cyclic succinimide intermediate (aminosuccinyl residue, Suc). These abnormal Asp residues are known to have relevance to aging and pathologies. Despite being non-enzymatic, the Suc formation is thought to require a catalyst under physiological conditions. In this study, we computationally investigated the mechanism of the Suc formation from Asp residues that were catalyzed by the dihydrogen phosphate ion, H2PO4−. We used Ac–l-Asp–NHMe (Ac = acetyl, NHMe = methylamino) as a model compound. The H2PO4− ion (as a catalyst) and two explicit water molecules (as solvent molecules stabilizing the negative charge) were included in the calculations. All of the calculations were performed by density functional theory with the B3LYP functional. We revealed a phosphate-catalyzed two-step mechanism (cyclization–dehydration) of the Suc formation, where the first step is predicted to be rate-determining. In both steps, the reaction involved a proton relay mediated by the H2PO4− ion. The calculated activation barrier for this mechanism (100.3 kJ mol−1) is in reasonable agreement with an experimental activation energy (107 kJ mol−1) for the Suc formation from an Asp-containing peptide in a phosphate buffer, supporting the catalytic mechanism of the H2PO4− ion that is revealed in this study.