Abstract
[7,3',5'-3H3]- and [7,9-3H3]-folic acid and [7,3',5'-3H3]methotrexate (MTX) have been prepared and 3H-n.m.r. spectra obtained for their complexes with Lactobacillus casei dihydrofolate reductase (DHFR). The 3H results confirm the presence of three pH-dependent different conformational forms in the complex DHFR.NADP+.folate. The folate benzoyl ring could be shown to be in essentially the same environment in the different forms, with the major differences being associated with the pterin ring. The appearance of a single resonance for the 3',5'-tritons showed that the benzoyl ring is flipping rapidly in all three forms. In contrast, the MTX complex was shown to exist as a single conformational state with the benzoyl ring flipping rate being too low to give a single averaged signal for the 3',5'-nuclei over the temperature range 283-313 K.