In proteins and peptides, d-aspartic acid (d-Asp) and d-β-Asp residues can be spontaneously formed via racemization of the succinimide intermediate formed from l-Asp and l-asparagine (l-Asn) residues. These biologically uncommon amino acid residues are known to have relevance to aging and pathologies. Although nonenzymatic, the succinimide racemization will not occur without a catalyst at room or biological temperature. In the present study, we computationally investigated the mechanism of succinimide racemization catalyzed by dihydrogen phosphate ion, H₂PO₄⁻, by B3LYP/6-31+G(d,p) density functional theory calculations, using a model compound in which an aminosuccinyl (Asu) residue is capped with acetyl (Ace) and NCH₃ (Nme) groups on the N- and C-termini, respectively (Ace–Asu–Nme). It was shown that an H₂PO₄⁻ ion can catalyze the enolization of the H_α–C_α–C=O portion of the Asu residue by acting as a proton-transfer mediator. The resulting complex between the enol form and H₂PO₄⁻ corresponds to a very flat intermediate region on the potential energy surface lying between the initial reactant complex and its mirror-image geometry. The calculated activation barrier (18.8 kcal·mol⁻¹ after corrections for the zero-point energy and the Gibbs energy of hydration) for the enolization was consistent with the experimental activation energies of Asp racemization.

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.

Spontaneous, nonenzymatic reactions in proteins are known to have relevance to aging and age-related diseases, such as cataract and Alzheimer’s disease. Among such reactions is the racemization of Ser residues, but its mechanism in vivo remains to be clarified. The most likely intermediate is an enol. Although being nonenzymatic, the enolization would need to be catalyzed to occur at a biologically relevant rate. In the present study, we computationally found plausible reaction pathways for the enolization of a Ser residue where a dihydrogen phosphate ion, H₂PO₄⁻, acts as a catalyst. The H₂PO₄⁻ ion mediates the proton transfer required for the enolization by acting simultaneously as both a general base and a general acid. Using the B3LYP density functional theory method, reaction pathways were located in the gas phase and hydration effects were evaluated by single-point calculations using the SM8 continuum model. The activation barriers calculated for the reaction pathways found were around 100 kJ mol⁻¹, which is consistent with spontaneous reactions occurring at physiological temperature. Our results are also consistent with experimental observations that Ser residue racemization occurs more readily in flexible regions in proteins.