Electrostatic interactions play an important role in enzyme catalysis. These effects are modulated by the conformational changes that occur over the catalytic cycle. To elucidate the catalytic roles of these effects, thiocyanate probes were introduced at site-specific positions in the enzymes ketosteroid isomerase (KSI) and dihydrofolate reductase (DHFR). In KSI, the impact of electrostatics on ligand binding was explored. In DHFR, the impact of electrostatics on the catalytic cycle involving five different complexes was investigated. The shifts in the vibrational stretching frequencies of the nitrile probes report on the electrostatics of the microenvironments surrounding the probes. Mixed quantum mechanical/molecular mechanical molecular dynamics simulations reproduced the experimental vibrational frequency shifts and provided atomic-level insight into the roles that key residues play in determining the electrostatics of the microenvironments. The electrostatic contributions were decomposed into the major components from individual residues, ligands, and water molecules. For DHFR, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motions occurring over the catalytic cycles of enzymes.