Abstract

Dihydrofolate reductase is of pharmacological interest as the target for a number of useful drugs including methotrexate, trimethoprim and pyrimethamine. The binding of substrates, inhibitors and coenzymes to the enzyme has been studied by NMR spectroscopy in conjunction with site-directed mutagenesis; changes in the chemical structure of the protein or the ligand are found to have a variety of effects on both the time-average conformation and its fluctuations. These experiments have revealed a number of instances where the specificity is influenced by conformational fluctuations or equilibria. Both the substrate, folate, and the coenzyme, NADP+, have alternative modes of binding to the enzyme. The pteridine ring of folate can bind in two distinct orientations, depending on the ionization state of Asp-26. In the enzyme-trimethoprim-NADP+ complex, the nicotinamide ring of the coenzyme exists in two states, either bound to the enzyme or hanging free in solution. Analogues of the inhibitor pyrimethamine exist in two slowly interconverting conformations, both of which are able to bind to the enzyme. In all these cases the different modes of ligand binding have very similar binding energies and play a role in determining the specificity of the enzyme.