Association of folate molecules as determined by proton NMR: Implications on enzyme binding

The self-association in aqueous solution of folic acid (FA), 7,8-dihydrofolic acid (DHFA) and 5,6,7,8-tetrahydrofolic acid (THFA) has been studied by the use of proton magnetic resonance ( 1H NMR) spectroscopy. At concentrations below 10 mM, all three folates exist in (monomer) 2⇌dimer equilibria with association constants (K a) equal to 400, 66 and 14 M −1 for FA, DHFA and THFA respectively. These values decreased markedly to 157, 18 and 3 M −1, for FA, DHFA and THFA respectively, in the presence of 0.8 M KCl. The high extent of dimerization of FA is believed to impede the interaction with the active site of dihydrofolate reductase (DHFR) rendering it a poor substrate. In contrast, the DHFA with a much lower K a is a better substrate. Conditions that lower the K a of both FA and DHFA, (i.e., 0.8M KCl) turn them into better substrates. Based on the findings of the present study, it is also predicted that dihydro MTX may be a better inhibitor of DHFR than MTX.