TY - JOUR
T1 - Evaluation of the Importance of Hydrophobic Interactions in Drug Binding to Dihydrofolate Reductase
AU - Taira, Kazunari
AU - Benkovic, Stephen J.
PY - 1988/1/1
Y1 - 1988/1/1
N2 - 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.
AB - 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.
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U2 - 10.1021/jm00396a019
DO - 10.1021/jm00396a019
M3 - Article
C2 - 3275776
AN - SCOPUS:0023860769
SN - 0022-2623
VL - 31
SP - 129
EP - 137
JO - Journal of Medicinal Chemistry
JF - Journal of Medicinal Chemistry
IS - 1
ER -