Structural comparisons of complexes of methotrexate analogues with Lactobacillus casei dihydrofolate reductase by two-dimensional 1H NMR at 500 MHz.

We have used two-dimensional (2D) NMR methods to examine complexes of Lactobacillus casei dihydrofolate reductase and methotrexate (MTX) analogues having structural modifications of the benzoyl ring [the 3',5'-difluoro and 3',5'-dichloro analogues (II and III)] and also the glutamic acid moiety [the alpha- and gamma-monoamides (IV and V)]. Assignments of the 1H signals in the spectra of the various complexes were made by comparison of their 2D spectra with those of complexes containing methotrexate where we have previously assigned resonances from 32 of the 162 amino acid residues. In the complexes formed with the dihalomethotrexate analogues, the glutamic acid and pteridine ring moieties were shown to bind to the enzyme in a manner similar to that found in the methotrexate-enzyme complex. Perturbations in 1H chemical shifts of protons in Phe-49, Leu-54, and Leu-27 and the methotrexate H7 and NMe protons were observed in the different complexes and were accounted for by changes in orientation of the benzoyl ring in the various complexes (15 degrees and 25 degrees in the difluoro- and dichloromethotrexate complexes, respectively). Binding of oxidized or reduced coenzyme (NADP+ or NADPH) to the binary complexes did not result in different shifts for Leu-27, Leu-54, or Leu-19 protons, and thus, the orientation of the benzoyl ring of the methotrexate analogues is not perturbed greatly by the presence of either oxidized or reduced coenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)