Abstract
Aspartic acid (Asp) residues in peptides and proteins are prone to isomerization to the β-form and racemization via a five-membered succinimide intermediate. These nonenzymatic reactions have relevance to aging and age-related diseases. In this paper, we report a three water molecule-assisted, six-step mechanism for the formation of succinimide from Asp residues found by density functional theory calculations. The first two steps constitute a stepwise iminolization of the C-terminal amide group. This iminolization involves a quintuple proton transfer along intramolecular and intermolecular hydrogen bonds formed by the C-terminal amide group, the side-chain carboxyl group, and the three water molecules. After a conformational change (which breaks the intramolecular hydrogen bond involving the iminol nitrogen) and a reorganization of water molecules, the iminol nitrogen nucleophilically attacks the carboxyl carbon of the Asp side chain to form a five-membered ring. This cyclization is accompanied by a triple proton transfer involving two water molecules, so that a gem-diol tetrahedral intermediate is formed. The last step is dehydration of the gem-diol group catalyzed by one water molecule, and this is the rate-determining step. The calculated overall activation barrier (26.7 kcal mol−1) agrees well with an experimental activation energy.
Highlights
Aspartic acid (Asp or D) residues in peptides and proteins (L-Asp) are prone to isomerization to the β-form and racemization under physiological conditions [1,2,3]
Note that SI is a complex including four water molecules because a water molecule is released in the last dehydration step
By density functional theory (DFT) calculations, we have been successful for the first time in showing a single reaction model which can qualitatively describe the entire multi-step process of succinimide formation from Asp residues
Summary
Aspartic acid (Asp or D) residues in peptides and proteins (L-Asp) are prone to isomerization to the β-form and racemization under physiological conditions [1,2,3] These reactions occur spontaneously (i.e., nonenzymatically) and interdependently via a cyclic intermediate (succinimide) as shown in Scheme 1, giving rise to the biologically uncommon L-β-Asp, D-Asp, and D-β-Asp residues. Our recent computational studies [11,12] have shown that, under physiological conditions, it is quite likely that the succinimide formation reactions from Asp residues are catalyzed by two or three water molecules. The first two steps, which constitute a stepwise iminolization process, involve a quintuple proton transfer along intramolecular and intermolecular hydrogen bonds This iminolization process is similar, except for the stepwise nature, to the previously reported two water molecule-catalyzed concerted mechanism [11]. The iminolization−cyclization−dehydration mechanism of the succinimide formation from an Asp residue
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