title: Evaluation of the importance of hydrophobic interactions in drug binding to dihydrofolate reductase.
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----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 (greater than 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.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802.
authors.forename: K
authors.initials: K
authors.lastname: Taira
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008727:Methotrexate / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011621:Pteridines / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D011621:Pteridines; C020280:2,4-diamino-6,7-dimethylpteridine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate