Determining nucleotide acidity and cation binding constants by ³¹P NMR

While the acid-base property of nucleotide phosphates and cation binding capacity can be determined by a variety of techniques, NMR spectroscopy has its unique advantage for providing site-specific information on these properties. Here we use Mg ²⁺- or Na ⁺-bound ATP to demonstrate how to deduce pK _a values for phosphate protonation - deprotonation and cation binding constants from ³¹P chemical shift data. Cation effects on nucleotide stability are also illustrated by a NMR kinetic study of myosin-catalyzed ATP hydrolysis. These NMR related analytical approaches are important for quantitatively exploring nucleotide-cation interaction and its effects on nucleotide conformations and functions under varied conditions.