Substrates reconfigure picosecond electric field fluctuations in dihydrofolate reductase

Enzymes dramatically accelerate reaction rates, yet the detailed mechanisms of enzymatic catalysis, particularly the role of rapid electrostatic fluctuations, remain elusive. This study investigates pico- and nanosecond dynamics within dihydrofolate reductase (DHFR) from E. coli, focusing on substrate effects on electric field fluctuations during catalysis. Using molecular dynamics (MD) simulations, we examined structural and electrostatic changes in ligand-bound (DHFR•NADP•FOL) and unbound (apoform) states. Results show that ligand binding increases structural flexibility and dynamic behavior, indicated by higher radius of gyration (Rg), accelerates the decay of the autocorrelation function at sub-picosecond timescales, while slowing it at longer timescales, and increases root mean square deviation (RMSD) in specific regions. We find substantially enhanced localized electric field fluctuations around certain residues in the ligand-bound form but not in the apoform indicating that ligand binding significantly alters the electrostatic environment during the catalysis and potentially on energy barrier crossing events. Our findings highlight the importance of structural and electrostatic dynamics in enzyme function. Insights from this study can be applied in the design of effective enzyme inhibitors and engineered enzymes, advancing synthetic biology and protein engineering development.