The nonenzymatic deamidation reactions of asparagine (Asn) and glutamine (Gln) residues in proteins are associated with protein turnover and age-related diseases. The reactions are also believed to provide a molecular clock for biological processes. Although Gln deamidation is assumed to occur through the glutarimide intermediate, the mechanisms for this are unclear because under normal physiological conditions, Gln deamidation occurs relatively less frequently and at a lower rate than Asn deamidation. We investigate the mechanisms underlying glutarimide formation from Gln residues, which proceeds in two steps (cyclization and deammoniation) catalyzed by phosphate and carbonate. We also compare these reactions with noncatalytic mechanisms and water-catalyzed mechanisms. The calculations were performed on the model compound Ace–Gln–Nme (Ace = acetyl, Nme = methylamino) using the density functional theory with the B3LYP/6-31+G(d,p) level of theory. Our results suggest that all the catalysts used in our study can mediate the proton relays required for glutarimide formation. We further determined that the calculated activation barriers of the reactions catalyzed by phosphate ions (115 kJ mol –1 ) and carbonate ions (112 kJ mol –1 ) are sufficiently low for the reactions to occur under normal physiological conditions. We also show that nucleophilic enhancement of Nme nitrogen is essential for the cyclization of Gln residues.

Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.

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Uncommon crosslinked amino acids have been identified in several aging tissues and are suspected to trigger various age-related diseases. Several uncommon residues are formed when the dehydroalanine (Dha) residue undergoes a nucleophilic attack by surrounding residues. Dha residues are considered to be formed by posttranslational modification of serine (Ser) and cysteine residues. In the present study, we investigated the Dha residue formation mechanism catalyzed by dihydrogen phosphate ion (H 2 PO 4 − ) using quantum chemical calculations. We obtained optimized geometries using the B3LYP density functional method and carried out single-point energy calculations using the second-order Møller−Plesset perturbation method. All calculations were performed using Ace-Ser-Nme (Ace = acetyl, Nme = methylamino) as a model compound. Results of the computational analysis suggest that the mechanism underlying the Dha residue formation from Ser consists of two steps: enolization and 1,3-elimination. The H 2 PO 4 − catalyzed both reactions as a proton-relay mediator. The calculated activation barrier for Dha residue formation was estimated as 30.4 kcal mol −1 . In this pathway, the catalytic H 2 PO 4 − interacts with the Ser residue α-proton, carbonyl oxygen of Ser, and C-terminal side adjacent residues, and the calculated activation energy produced was the same as the experimentally reported value for nonenzymatic modifications of amino acid residues. Therefore, our calculation suggests that H 2 PO 4 − -catalyzed Ser residue dehydration can proceed nonenzymatically.