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).

A 1H n.m.r. study of the role of the glutamate moiety in the binding of methotrexate to Lactobacillus casei dihydrofolate reductase

“…The decrease in pK of His 18 accompanying methotrexate binding (Table 3) appears to reflect an inhibitor-induced conformational change (Birdsall et al, 1977;Wyeth et al, 1980;Gronenbom et al, 1981); this decrease in pK appears to be essentially the same for the a-and y-amides as for methotrexate itself. However, significantly larger decreases are observed for both the a,y-diamide and the a,y-dimethyl ester, indicating a significantly different mode of binding.…”

“…(Birdsall et al, 1977;Wyeth et al, 1980;Gronenborn et al, 1981), and assignments for all their C2-proton resonances are now available (Wyeth et al, 1980; Birdsall, J. Feeney and G.C.K. Roberts, unpublished work), although some of these remain tentative.…”

Potentiating effect of pyrimethamine and sulfadoxine against dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi

“…Fluorometric measurements of the binding of 2,4-diaminopyrimidine and p-aminobenzoyl-L-glutamate to Lactobacillus casei cells revealed that the two fragments bind the bacterial enzyme simultaneously and cooperatively (5,6). Moreover, a recent study concerning the in vitro evaluation of the antimalarial activity of pyrimethamine and sulfadoxine against Plasmodium falciparum also suggested the simultaneous-inhibition hypothesis as a possible mechanism of potentiation (13).…”

“…Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3), which catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, has been regarded as one of the crucial targets for bacterial and protozoal chemotherapy. The enzyme was shown to be the molecular site of action of a number of antifolates (2,8,32).…”

NMR Studies of Ligand Binding to Dihydrofolate Reductase

“…1 pK unit higher than its value in the ligand-free enzyme ( Figure 17) [130,131] This value reflects the interaction of His 28 with the MTX g-carboxylate group ( Figure 13) and provides an excellent method for monitoring interactions involving His 28. [132] We have used these effects to study complexes of L. casei DHFR with the a-and g-amide (5); (!)…”