1H-NMR Studies on Nucleotide Binding to the Sarcoplasmic Reticulum Ca2+ ATPase

The glycosidic bond torsion angles and the conformations of the ribose of Mg2+ ATP, Mg2+ ADP and Mg2+ AdoPP[NH]P (magnesium adenosine 5′-[β,γ-imido]triphosphate) bound to Ca2+ ATPase, both native and modified with fluorescein isothiocyanate (FITC), in intact sarcoplasmic reticulum have been determined by the measurement of proton-proton transferred nuclear Overhauser enhancements by 1H-NMR spectroscopy. This method shows clearly the existence of a low-affinity ATP binding site after modification of the high-affinity site with FITC. For all three nucleotides bound to both the high-affinity (catalytic) site and the low-affinity site, we find that the conformation about the glycosidic bond is anti, the conformation of the ribose 3′-endo of the N type and the conformation about the ribose C4′-C5′ bond either gauche-trans or trans-gauche. The values for the glycosidic bond torsion angles χ (O4′-C1′-N9-C4) for Mg2+ ATP, Mg2+ ADP and Mg2+ AdoPP[NH]P bound to the low-affinity site of FITC-modified Ca2+ATPase are ∼ 270°, ∼ 260° and ∼ 240° respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ATPase, χ lies in the range 240–280°.