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 → Tyr) has a minimal effect on the binding of the classical inhibitor, DAM. On the other hand, the second mutation (Phe-31 → 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.
All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Drug Discovery