Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to lack of experimental access. This shortcoming is evident with E. coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we present ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments that enable identification of coupled conformational changes in DHFR. We identify a global hinge motion and local networks of structural rearrangements that are engaged by substrate protonation to regulate solvent access and promote efficient catalysis. The resulting mechanism shows that DHFR's two-step catalytic mechanism is guided by a dynamic free energy landscape responsive to the state of the substrate.

Resolving conformational changes that mediate a two-step catalytic mechanism in a model enzyme