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 Lactobacillus casei dihydrofolate reductase on the histidine residues of the enzyme have been studied by 1 H n. m. r. spectroscopy. The more weakly binding (rapidly exchanging) inhibitors 2, 4-diaminopyrimidine and p -aminobenzoyl-l-glutamate, which can be regarded as ‘fragments’ of methotrexate, have also been studied as an aid in interpreting the effects of the strongly-binding ligands. p -Aminobenzoyl-l-glutamate binds to two sites on the enzyme; binding to the stronger site is competitive with methotrexate, and affects three histidine residues, denoted H A , H E and H F . The second site is 30-fold weaker, is not competitive with methotrexate, and affects a single histidine residue (either H B or H C ). The binding to the first site is 25-fold stronger in the presence of 2, 4-diaminopyrimidine, while binding to the second site is unaffected. Folate, dihydrofolate and folinic acid have identical effects on the histidine residues; the p K of H E is increased from 6.54 to 6.75, and that of H F from 6.54 to ca . 7.2, while the C2-H resonance of H A is shifted downfield. Methotrexate and amino­pterin affect the same three histidine residues as does folate; for H A and H F the effects are the same as those produced by folate, while the p K of H E 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 H F is not affected by these compounds (which lack the p -amino-benzoyl-l-glutamate moiety).