Dihydrofolate Reductase from a Methotrexate-resistant Escherichia coli

Upon binding of pyridine nucleotides to Escherichia coli MB 1428 dihydrofolate reductase, absorption bands of both enzyme and pyridine nucleotide are changed. Titration of enzyme with TPNH confirms the presence of two TPNH binding sites. Similar titrations show two binding sites for TPNH on a 1:1 complex of enzyme with methotrexate, two sites for TPN+ on both enzyme and a 1:1 enzyme-methotrexate complex, and at least one DPNH binding site on the enzyme-methotrexate complex. Equilibrium ultrafiltration measurements confirm the presence of two TPN+ binding sites on both enzyme and 1:1 enzyme-methotrexate and demonstrate the presence of two DPNH binding sites on 1:1 enzyme-methotrexate. Titrations and comparison of 2:1 with 1:1 pyridine nucleotide-enzyme difference spectra reveal that most of the absorbance changes associated with binding occur upon binding at the higher affinity site. Dissociation constants for TPN+ and DPNH were calculated from these titrations: E·(TPN+)2, 80 ± 40 µm and 5 ± 3 µm; E·MTX·(TPN+)2, 1.7 ± 0.9 µm and 0.06 ± 0.03 µm; E·MTX·(DPNH)2, 100 ± 50 µm and 9 ± 5 µm. Absorbance changes at 280 and 290 nm suggest the involvement of at least 1 tryptophan residue in the binding of TPNH, and possibly TPN+, at the higher affinity site. Absorbance changes upon binding of DPNH, TPNH, and TPN+ to the enzyme-methotrexate complex indicate that all three cofactors are bound identically. The binding of pyridine nucleotides to the enzyme-methotrexate complex changes the absorption bands of methotrexate toward an even more acidic spectrum.