Recently, two sets of single, double, and quadruple residue changes within the hydrophobic substrate binding pocket of Escherichia coli dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 18.104.22.168) were shown to exhibit nonadditive mutational effects [Huang, Z., Wagner, C. R., & Benkovic, S. J. (1994) Biochemistry 33, 11576-11585]. In particular, the analysis of data for the L28Y, L54F, and L28Y-L54F mutations revealed nonadditive changes in the free energy associated with the substrate and cofactor binding, hydride transfer, and product release steps. Construction of a related set of mutant proteins including L28F and L28F-L54F permits a comparison of similar energy changes and provides a means for assessing differences in the interactions of Phe28 and Tyr28 with both the ligands and the side chains at residue 54. We find a single functional group change, from Phe C4-H to Tyr C4-OH, can influence the additivity of mutational effects and serve as a probe to monitor the appearance of differing enzyme conformations along the reaction pathway through changes in the interaction energy (∆G1). The comparison of additivity/nonadditivity in free energy changes for three interrelated double mutational cycles (WT→ L28F-L54F, WT→L28Y-L54F, and L28F→ L28Y-L54F) demonstrates that the side chains of positions 28 and 54 interact cooperatively to facilitate hydride transfer by preferentially influencing the enzyme-substrate ground-state complexes. The AGi data for individual steps also provide evidence for multiple conformations of the enzyme operating during the catalytic cycle. The fact that there are no published examples of the synergistic enhancement of favorable mutational effects is consistent with the expectation that the binding/active site surface of wild-type dihydrofolate reductase has been optimized.