Evaluation of the importance of hydrophobic interactions in drug binding to dihydrofolate reductase

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Abstract
The interaction of dihydrofolate reductase (DHFR) from Escherichia coli with drugs such as methotrexate (MTX) and 2,4-diamino-6,7-dimethylpteridine (DAM) has been studied by means of site-directed mutagenesis, fluorescence spectroscopy, and steady-state as well as transient kinetics. A strictly conserved residue at the dihydrofolate binding site of DHFR, phenylalanine-31, has been replaced with tyrosine or valine to ascertain the importance for binding of this hydrophobic amino acid, which interacts with both the pteridine ring and the p-aminobenzoyl moiety. The first mutation (Phe-31 .fwdarw. Tyr) has a minimal effect on the binding of the classical inhibitor, DAM. On the other hand, the second mutation (Phe-31 .fwdarw. Val) has increased the dissociation constant of DAM from the DHFR-NADPH-DAM ternary complex over 150-fold (> 3 kcal/mol). The dissociation constant of DAM from the (Val31-DHFR)-DAM binary complex was too large to be measured fluorometrically. More importantly, these mutations have decreased the overall tight binding of MTX, from 100- to 140-fold (corresponding to a loss of binding energy of 2.2-2.4 kcal/mol) for the Tyr-31 and Val-31 mutants, respectively. These results indicate that hydrophobic interactions between MTX and DHFR are at least as important as formation of the MTX-DHFR salt bridge in the tight binding of MTX.