The variable residue Leu-28 of Escherichia coli dihydrofolate reductase (DHFR) and the corresponding residue Phe-31 in murine DHFR were interchanged, and the impact on catalysis was evaluated by steady-state and pre-steady-state analysis. The E. coli L28F mutant increased the pH-independent Kcat from 11 to 50 s-1but had little effect on Km(H2F). An increase in the rate constant for dissociation of H4F from E-H4F-NH (from 12 to 80 s-1) was found to be largely responsible for the increase in kcat. Unexpectedly, the rate constant for hydride transfer increased from 950 to 4000 s-1with little perturbation of NADPH and NADP+binding to E. Consequently, the flux efficiency of the E. coli L28F mutant rose from 15% to 48% and suggests a role in genetic selection for this variable side chain. The murine F31L mutant decreased the pH-independent Kcat from 28 to 4.8 s-1but had little effect on Km(H2F). A decrease in the rate constant for dissociation of H4F from E-H4F-NH (from 40 to 22 s-1) and E-H4F (from 15 to 0.4 s-1) was found to be mainly responsible for the decrease in k. The rate constant for hydride transfer decreased from 9000 to 5000 s-1with minor perturbation of NADPH binding. Thus, the free energy differences along the kinetic pathway were generally similar in magnitude but opposite in direction to those incurred by the E. coli L28F mutant. This conclusion implies that DHFR hydrophobic active-site side chains impart their characteristics individually and not collectively.