A conserved residue at the dihydrofolate binding site of dihydrofolate reductase (EC 1.5.1.3), leucine-54, was replaced with glycine to ascertain the role of this hydrophobic amino acid. The effect of the mutation is both to increase the dissociation rate of dihydrofolate and decrease the rate of hydride transfer thus changing the rate-limiting step in catalysis from product loss (leucine-54) to hydride transfer (glycine-54). The total stabilization by leucine-54 of the transition state for hydride transfer is ca. 10(4)-fold (delta delta G approximately 5.4 kcal/mol) at subsaturating dihydrofolate levels relative to free enzyme despite its location some 10 A from the site of chemical reaction.