Deamidation of Asparagine Residues: Direct Hydrolysis versus Succinimide-Mediated Deamidation Mechanisms

Quantum chemical calculations are reported to provide new insights on plausible mechanisms leading to the deamidation of asparagine residues in proteins and peptides. Direct hydrolysis to aspartic acid and several succinimide-mediated mechanisms have been described. The catalytic effect of water molecules has been explicitly analyzed. Calculations have been carried out at the density functional level (B3LYP/6-31+G**). Comparisons of free energy profiles show that the most favorable reaction mechanism goes through formation of a succinimide intermediate and involves tautomerization of the asparagine amide to the corresponding imidic acid as the initial reaction step. Another striking result is that direct water-assisted hydrolysis is competitive with the succinimide-mediated deamidation routes even in the absence of acid or base catalysis. The rate-determining step for the formation of the succinimide intermediate is cyclization, regardless of the mechanism. The rate-determining step for the complete deamidation is the hydrolysis of the succinimide intermediate. These results allow clarification of some well-known facts, such as the isolation of succinimide or the absence of iso-Asp among the reaction products observed in some experiments.