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. 104-fold (AAG 5.4 kcal/mol) at subsaturating dihydrofolate levels relative to free enzyme despite its location some 10 A from the site of chemical reaction.Classical mechanistic studies on enzyme catalysis have focused on active site residues involved as acid-base catalysts or in the formation of covalent intermediates. The importance of hydrophobic amino acids remote from or hydrophobic residues directly at the active site could not be readily ascertained. Site-directed mutagenesis provides a means to define the role of hydrophobic amino acids in enzymic catalysis. To date, the focus of most studies using site-directed mutagenesis remains on active site residues, deleting them or replacing amino acids with potentially more efficient catalysts (1-3). One striking mutagenesis study on t-RNATYr ligase (formerly t-RNATYr synthetase) (4) suggests the critical importance of remote amino acids in catalysis. Replacing two hydrogen-bonding residues in the binding site for the y-phosphate of ATP (nucleophilic attack occurs at the aPhosphate) decreased the rate of catalysis by a factor of 10 , while only weakening ATP binding by a factor of 5. We report here the effect on catalysis and binding of a mutation in dihydrofolate reductase (DHFR; EC 1.5. MATERIALS AND METHODSThe Gly-54 and Val-31 mutants were constructed by primer extension of oligonucleotides using as the template partially single-stranded pTY1 (8, 9), a derivative of pBR322 containing a 1-kilobase insert encoding forE. coli DHFR. The double mutant was constructed in the same manner, but using the Val-31 encoding plasmid as the template. The oligonucleotide sequence introduced a new restriction site (Fig. 1) 7718The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.