Possible Mechanisms of Nonenzymatic Formation of Dehydroalanine Residue Catalyzed by Dihydrogen Phosphate Ion

Abstract

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.

Predicting Reaction Mechanisms for the Threonine-Residue Stereoinversion Catalyzed by a Dihydrogen Phosphate Ion

“… 24 In addition, our recent computational studies showed that the activation energies of Ser-residue dehydration and Dha-residue hydration were significantly higher than that of Ser-residue enolization. 25 These data supported that the Ser-residue stereoinversion mainly proceeds by keto-enol tautomerization. l -Ala- d / l -Glu epimerase is an enzyme that catalyzes keto-enol tautomerization with proton abstraction.…”

“…Previously, quantum chemical calculations showed that the activation energies of Ser-residue stereoinversion via enol intermediates are comparable to those of typical nonenzymatic reactions. 25 , 27 Although Dha residues are potential intermediates of Ser-residue stereoinversion, experimental and computational evidence so far has confirmed the hypothesis that Dha residues are intermediates of stereoinversion. 24 , 25 Therefore, in the present study, d - allo -Thr residue was assumed to be obtained via enolized Thr residues ( Scheme 3 ), as well as Ser-residue stereoinversion.…”

“… 25 , 27 Although Dha residues are potential intermediates of Ser-residue stereoinversion, experimental and computational evidence so far has confirmed the hypothesis that Dha residues are intermediates of stereoinversion. 24 , 25 Therefore, in the present study, d - allo -Thr residue was assumed to be obtained via enolized Thr residues ( Scheme 3 ), as well as Ser-residue stereoinversion. In previous computational studies, it has been shown that the calculated activation energies for several nonenzymatic modifications of amino acid residues in the absence of any catalysts are too high to proceed under physiological conditions.…”

“…Previous computational studies showed that a dihydrogen phosphate ion (H 2 PO 4 – ) could catalyze several nonenzymatic modifications of amino acid residues. 25 , 27 , 31 − 40 In these studies, H 2 PO 4 – acts as both a Brønsted acid and base, and the catalyzed proton relays after forming appropriate complexes with amino acid residues. In the present study, to investigate why Thr-residue stereoinversion has not been observed in vivo, computational analysis was performed using quantum chemical calculations to elucidate the mechanisms for the stereoinversion of Thr residues catalyzed by H 2 PO 4 – .…”

Computational Studies on Water-Catalyzed Mechanisms for Stereoinversion of Glutarimide Intermediates Formed from Glutamic Acid Residues in Aqueous Phase

“…Recently, we performed benchmark studies on the mechanism of nonenzymatic Asp-residue cyclization, and showed that the calculations using B3LYP functional methods provided favorable results [34]. In addition, the activation energies for non-enzymatic modifications of amino acid residues were successfully calculated using the B3LYP/6-31+G(d,p) level of theory [23,24,25,26,27,28,29,30,35,36]. Thus, we used B3LYP/6-31+G(d,p) for this study.…”