Methotrexate binding to dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli.

Activity and fluorescence studies have been performed on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B. Whereas the activity of this enzyme does not depend on the sequence of addition of substrate and cofactor, inhibition by methotrexate has been found to be dependent on preincubation conditions, as well as on ionic strength and dihydrofolate concentration. The enzyme exhibits turnover numbers of 1050 f 50 moles of dihydrofolate reduced per min per mole of methotrexate binding sites on the enzyme in 0.05 M Tris-HCl buffer, pH 7.2 at ‘25”, and 650 3t 50 in 0.05 M Tris-HCl-0.05 M NaCl, pH 7.2 at 25”. Methotrexate inhibition was found to be competitive, with an apparent Ki of 0.36 nM when the enzyme is preincubated with methotrexate, and 2.4 IIM when preincubated with methotrexate and an excess of dihydrofolate. When the enzyme is preincubated with reduced triphosphopyridine nucleotide and methotrexate, the inhibition is essentially stoichiometric and thus only an upper limit of 0.01 nM for Ki can be determined. The dissociation constants for the enzyme-methotrexate complexes were estimated from fluorometric measurements and ranged from 1 to 27 nM as a function of ionic strength.