Towards the Accurate Thermodynamic Characterization of Enzyme Reaction Mechanisms

We employed QM/MM molecular dynamics (MD) simulations to characterize the rate-limiting step of the glycosylation reaction of pancreatic alpha-amylase with combined DFT/molecular dynamics methods (PBE/def2-SVP:AMBER). Upon careful choice of four starting active site conformations based on thorough reactivity criteria, Gibbs energy profiles were calculated with umbrella sampling simulations within a statistical convergence of 1-2 kcal.mol(-1). Nevertheless, Gibbs activation barriers and reaction energies still varied from 11.0 to 16.8 kcal.mol(-1) and -6.3 to +3.8 kcal.mol(-1) depending on the starting conformations, showing that despite significant state-of-the-art QM/MM MD sampling (0.5 ns/profile) the result still depends on the starting structure. The results supported the one step dissociative mechanism of Asp197 glycosylation preceded by an acid-base reaction by the Glu233, which are qualitatively similar to those from multi-PES QM/MM studies, and thus support the use of the latter to determine enzyme reaction mechanisms.

Automated summary

In this study, QM/MM molecular dynamics simulations were used to investigate the rate-limiting step of the glycosylation reaction of pancreatic alpha-amylase. By carefully selecting different starting conformations and performing umbrella sampling simulations, Gibbs energy profiles were calculated. The results showed that the starting conformation has a significant impact on the reaction energy and activation energy.