Use of transferred nuclear Overhauser effect measurements to compare binding of coenzyme analogs to dihydrofolate reductase

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Abstract
Transferred nuclear Overhauser effect measurements were made on complexes of NADP+ and thioNADP+ with Lactobacillus casei dihydrofolate reductase to provide information about the glycosidic bond conformations in these complexes. NADP+ and thioNADP+ have very similar anti conformations about their adenine glycosidic bonds when bound to the enzyme, but their nicotinamide glycosidic bond conformations are very different; while NADP+ binds in an exclusively anti conformation, thioNADP+ binds with a distribution of syn/anti conformations very similar to that observed in nicotinamide mononucleotides in free solution (.apprx. 50:50). For thioNADP+, binding to the enzyme does not significantly perturb the potential function for rotation about the nicotinamide glycosidic bond. Earlier NMR studies indicated that large downfield 1H shifts of the nicotinamide ring protons (0.61-1.36 ppm) are detected on binding NADP+, while only very small shifts (< 0.1 ppm) are observed in complexes with thioNADP+. The chemical shift and conformational findings are best explained if the thionicotinamide ring extends into solution making essentially no contacts with the enzyme.

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