1 H nuclear magnetic resonance studies of Lactobacillus casei dihydrofolate reductase: effects of substrate and inhibitor binding on the histidine residues

Abstract

The effects of the binding of substrates and inhibitors (folate, dihydrofolate, folinic acid, trimethoprim, methotrexate and aminopterin) to L. casei dihydrofolate reductase [EC 1.5.1.3] on the histidine residues of the enzyme were studied by PMR spectroscopy. The more weakly binding (rapidly exchanging) inhibitors 2,4-diaminopyrimidine and p-aminobenzoyl-L-glutamate, which can be regarded as fragments of methotrexate, were studied as an aid in interpreting the effects of the strongly-binding ligands. p-Aminobenzoyl-L-glutamate binds to 2 sites on the enzyme; binding to the stronger site is competitive with methotrexate and affects 3 histidine residues, denoted HA, HE and HF. The 2nd site is 30-fold weaker, is not competitive with methotrexate and affects a single histidine residue (either HB or HC). The binding to the 1st site is 25-fold stronger in the presence of 2,4-diaminopyrimidine, while binding to the 2nd site is unaffected. Folate, dihydrofolate and folinic acid have identical effects on the histidine residues; the pK of HE is increased from 6.54 to 6.75, and that of HF from 6.54 to approximately 7.2, while the C2-H resonance of HA is shifted downfield. Methotrexate and aminopterin affect the same 3 histidine residues as does folate; for HA and HF the effects are the same as those produced by folate, while the pK of HE is decreased from 6.54 to 6.2. Trimethoprim and 2,4-diaminopyrimidine have effects very similar to those of methotrexate, with the exception that histidine HF is not affected by these compounds (which lack the p-aminobenzoyl-L-glutamate moiety).