Acetic acid can catalyze succinimide formation from aspartic acid residues by a concerted bond reorganization mechanism: a computational study.

Ohgi Takahashi,Ryota Kirikoshi,Noriyoshi Manabe

doi:10.3390/ijms16011613

Ohgi Takahashi, Ryota Kirikoshi + Show 1 more

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https://doi.org/10.3390/ijms16011613

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Abstract

Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism.

Highlights

Among nonenzymatic post-translational modifications of proteins, the alterations of normal L-aspartic acid (L-Asp) residues to L-β-Asp, D-Asp and D-β-Asp residues have recently attracted considerable attention because of relevance to aging and pathologies [1,2,3,4,5,6,7,8,9,10,11,12]
We computationally show that an acetic acid (AA) molecule (CH3COOH, not CH3COO−) can catalyze the two-step formation of succinimide from L-Asp residues (Scheme 2)
While geometry optimizations and zero-point energy (ZPE) calculations were performed in a vacuum, hydration free energies estimated by the SM8 continuum model [56,57] were taken into account in relative energy calculations

Summary

Introduction

Among nonenzymatic post-translational modifications of proteins, the alterations of normal L-aspartic acid (L-Asp) residues to L-β-Asp, D-Asp and D-β-Asp residues have recently attracted considerable attention because of relevance to aging and pathologies (especially those of age-related diseases, such as cataract and Alzheimer’s disease) [1,2,3,4,5,6,7,8,9,10,11,12] These altered Asp residues are formed through a five-membered cyclic succinimide intermediate having an aminosuccinyl (Asu) residue instead of Asp (Scheme 1) [13,14]. The first step is an intramolecular addition (cyclization) in which the amide nitrogen of the C-terminal peptide bond nucleophilically attacks the carboxyl carbon of the Asp side chain This gives a tetrahedral intermediate, which is probably a gem-diol at neutral to acidic pH [20,21]. Asp residues exist essentially in the deprotonated form (–COO−) at neutral or physiological pH, the amount of the protonated form increases as pH decreases

Results and Discussion

Computational Details

Conclusions