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Proton nmr and phosphorus 31 nmr characterization of 2 conformations of the trimethoprim nadp di hydro folate reductase complex



Proton nmr and phosphorus 31 nmr characterization of 2 conformations of the trimethoprim nadp di hydro folate reductase complex



Molecular Pharmacology 20(1): 145-153



The ternary complex between Lactobacillus casei dihydrofolate reductase, the coenzyme NADP+ and the antibacterial drug trimethoprim was studied by 1H and 31P NMR spectroscopy. The C2-H resonances of 2 of the histidine residues of the protein were each split into 2 signals of approximately half-proton intensity in the 1H spectrum of this complex. Studies of the temperature-dependence of the lineshape of these histidine signals showed that the splitting is due to the coexistence of approximately equal amounts of 2 slowly interconverting (6 s-1 at 31.degree. C) conformation forms of the complex. Two sets of proton resonances from the bound coenzyme were identified by the use of selectively deuterated coenzyme and by transfer of saturation experiments. Conformation I was characterized by nicotinamide proton resonances shifted substantially (0.6-1.1 ppm) to low field from their positions in the free coenzyme, while in Conformation II the changes in chemical shift on binding were much smaller (.ltoreq. 0.12 ppm). Only a single set of 1H resonances from the bound trimethoprim was observed in transfer of saturation experiments at 45.degree. C, perhaps because of relatively rapid interconversion between the 2 conformational states at this temperature. The addition of NADP+ produced a large (1.1 ppm) upfield shift of the 2',6'-proton resonance of trimethoprim relative to its position in the binary complex. In the 31P spectrum of the bound coenzyme, 2 sets of signals were seen for the pyrophosphate phosphorous nuclei. As judged from both the 1H and 31P spectra, the complexes of enzyme, trimethoprim and NADP+ or NHDP+ (the hypoxanthine analog) are mixtures of Conformations I and II, whereas the complexes formed with the thionicotinamide or acetylpyridine analogs of the coenzymes (TNADP+ and APADP+) are exclusively in Conformation II. The enzyme-methotrexate-NADP+ complex is exclusively in Conformation I. Comparing the 31P spectra of the enzyme-trimethoprim-TNADP+ and enzyme-methotrexate-NADP+ complexes, the 2 conformation states differed in the conformation of the pyrophosphate backbone of the bound coenzyme, as indicated by the 31P-1H and 31P-31P spin-spin coupling constants. This system appears to be an example of a 2-state conformational equilibrium which can be switched by the binding of ligands of different structure. The nature of the 2 conformational states and their implications for structure-activity analysis are discussed.

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Accession: 006215671

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