A Combined Experimental-Theoretical Approach Unveils the Comprehensive Asn Deamidation Kinetics.

The mechanism of the spontaneous deamidation reaction of the Asn residue in solution has been disclosed through a combination of experimental and computational methods. Utilizing the dipeptide Ace-AsnGly-NH2 (AsnGly) as a model system, the free energy barriers of the reaction steps have been modeled in parallel with time-resolved measurements of all species involved, obtained by NMR and IR spectroscopy. The convergent results of the theoretical and experimental data provide a quantitative description of all the kinetic constants for this four-step reaction. Furthermore, this study lends support to the two-step mechanism hypothesis for succinimide formation proposed in the literature and untangles the molecular origin of the reaction's pH dependence. By bridging atomic structure to reaction kinetics, this work paves the way for the next-generation prediction algorithms, which are essential for the advancement of biologics.