We have calculated the distribution of activation energies for the key hydride transfer step in the reduction of dihydrofolate by the enzyme dihydrofolate reductase in wild-type DHFR as well as the G121S and G121V variants of the Escherichia coli enzyme using combined quantum mechanical/molecular mechanical methods. Comparison of the activation energy distributions present in the three protein systems demonstrates that the ensemble of energy barriers differs in each. The features of these energy barrier distributions are consistent with experimentally determined reaction rates for the three proteins and support experimental observations demonstrating time-dependent variation in the reaction rate of individual enzyme molecules. This occurrence suggests that several distinct conformational substates in the proteins modify the potential energy surface for the reaction. A select set of distances and dihedral angles was observed to correlate well with the progress of the reaction or with the presence of protein conformations giving rise to low reaction barriers. These geometric parameters highlight features in the wild-type and mutant conformational ensembles that may give rise to the observed energy distributions. Our work demonstrates that these structural probes are indicators of changes in the equilibrium properties of the enzyme systems studied; they do not appear to arise as the result of direct coupling of time-dependent protein displacements to the reactive event.