1H and 19F NMR signals from bound ligands have been assigned in one-and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4′-chlorophenyl)-6-ethylpyrimidine], fluoro-pyrimethamine [2, 2,4-diamino-5-(4′-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4′-fluoro-3′-nitrophenyl)-6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4′-(methylbenzylamino)-3′-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from ~ 180° rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein. Ternary complexes with NADP+ were also examined: in the case of pyrimethamine and fluoronitropyrimethamine, conformations I and II, similar to those previously observed for enzyme complexes with trimethoprim and NADP+, were detected in the 31P spectra. The NADP+ nicotinamide ring in form I binds within the protein with its glycosidic bond fixed in an anti conformation, while in form II the conformation of the pyrophosphate moiety is altered so that the nicotinamide ring extends away from the protein in a mixture of syn and anti conformations [Birdsall, B., Bevan, A. W., Pascual C., Roberts, G. C. K., Feeney, J., Gronenborn, A., & Clore, G. M. (1984) Biochemistry 23, 4733-4742]. For fluoronitropyrimethamine the conformational preference for binding is reversed in the binary and ternary complexes (the A/B ratio being 0.6/0.4 in the binary and 0.3/0.7 in the ternary complex). This is consistent with a model in which the conformational forms are correlated, with only forms IB and IIA being populated: the NO2 substituent is oriented away from the nicotinamide ring binding site in form IB and toward the vacant site for nicotinamide ring binding in form IIA. Such a simple correlation cannot exist for the ternary complex of the enzyme with methylbenzoprim and NADP+, which exists exclusively in form I but has both forms A and B significantly populated: when the preference for form I is high the correlation between forms A/B and I/II is less important. © 1990, American Chemical Society. All rights reserved.
