BPS2026 – Interplay of electrostatics and conformational dynamics in dihydrofolate reductase catalysis

Electrostatic stabilization of transition states remains a central hypothesis for explaining the remarkable efficiency of enzymes. Less understood is how protein conformational dynamics affect electrostatic preorganization in the enzyme active site. The NADPH-coupled enzyme E. coli dihydrofolate reductase (ecDHFR) is highly flexible, while the substantial charge movement involved in ecDHFR-catalyzed hydride transfer suggests a catalytic role of electrostatics. We measured these electrostatic influences by replacing the transferable hydride on NADPH with a deuterium to generate a carbon-deuterium (C-D) vibrational Stark probe, precisely calibrated to measure electric fields spectroscopically. We then measured electric fields along the C-D axis approximating the reaction coordinate in purified ecDHFR with NADP( 2 H) and folate and its various analogues. In a reactive ternary complex with bound folate, the C-D infrared spectrum can be deconvolved into two populations suggesting a red-shifted electrostatically preorganized and a blue-shifted non-preorganized state. The populations undergo a temperature-dependent exchange over 283K–323K, possibly coinciding with movement in the M20 loop observed in recent temperature-dependent X-ray crystallographic studies over the same temperature range. 1 The enthalpy difference of these populations (∼24 kJ/mol) is similar to the activation energy of the hydride transfer reaction. The conformational change is supported by fixed-charge and polarizable molecular dynamics simulations, which show that ecDHFR’s M20 side chain regulates solvent access to the active site, enabling successive substrate protonation and hydride transfer within the same catalytic active site. Moreover, we note how substrate modifications or environmental mutations to residues M20 and P21 modulate the equilibrium between electrostatic states, observed in both the spectroscopic results and crystallographic structures. Our results suggest a complementary mechanism by which electrostatics and conformational dynamics together influence enzyme-catalyzed reactions.