Evidence for Two Reduced Triphosphopyridine Nucleotide Binding Sites on Dihydrofolate Reductase from a Methotrexate-resistant Strain of Escherichia coli

Fluorescence and ultrafiltration experiments were performed on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B. These studies demonstrate two reduced triphosphopyridine nucleotide binding sites on the enzyme. When TPNH is bound to dihydrofolate reductase, the enzyme fluorescence is partially quenched and the fluorescence of the bound TPNH is enhanced. The degree of quenching and enhancement is a function of the ionic strength of the buffer. The dissociation constants at pH 7.2 are 0.02 ± 0.01 µm and 0.6 ± 0.1 µm in a buffer of 0.10 m ionic strength, 0.2 ± 0.1 µm and 0.4 ± 0.3 µm in a solution of 0.35 m ionic strength, and 0.3 ± 0.2 µm and 4.0 ± 2.0 µm in 0.55 m ionic strength. The results demonstrate that one of the sites increases its affinity relative to the other in solutions of low ionic strength. Ultrafiltration experiments indicate that the enzyme has two TPNH binding sites in the presence of methotrexate, but fluorometric experiments detect the binding of only 1 mole of TPNH per mole of enzyme. The binding of the 2nd mole of TPNH to the enzyme-methotrexate-TPNH complex does not perturb the TPNH fluorescence. The affinity of the enzyme-TPNH complex for methotrexate is greater than that of the enzyme alone.