Interaction of methotrexate metabolites with beef liver dihydrofolate reductase-I. Binary complex study.

In order to explain the difference of inhibition of dihydrofolate reductase (DHFR) by methotrexate (MTX) and its metabolites 7-hydroxymethotrexate [7OH (MTX)] and polyglutamate derivatives [MTX (G1) and MTX (G2)], direct determinations of binding parameters to beef liver DHFR were performed. Association constants are calculated by fluorescence titrations and thermodynamic parameters by microcalorimetric measurements. The parameters of interaction are nearly identical for MTX and polyglutamate derivatives but are different for MTX and 7OH (MTX). For this last derivative electrostatic forces are less predominant and a larger modification of its conformation appears in the enzyme during the complex formation.