Phosphorus-31 nuclear magnetic resonance studies of the binding of oxidized coenzymes to Lactobacillus casei dihydrofolate reductase

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Abstract
The 31P NMR spectra of NADP+ and a number of its structural analogs were obtained from their binary and ternary complexes with L. casei dihydrofolate reductase. The 2''-phosphate resonance is shifted downfield 2.7-2.9 ppm in all cases. Line-shape analysis of this resonance as a function of coenzyme concentration gave values for the dissociation rate constant of the coenzyme from many of the complexes. The values obtained are discussed in terms of the kinetic mechanism of coenzyme binding. The chemical shifts of the pyrophosphate resonances vary from 1 complex to another over a range of 3.8 ppm. The assignment of these signals to the individual pyrophosphate 31P nuclei and the structural origins of the chemical shift changes are discussed. These data and the 1H NMR experiments described previously showed that the nicotinamide end of the thionicotinamide and acetylpyridine coenzyme analog binds to the enzyme quite differently from that of the natural coenzyme NADP+.