Determination of the Conformations of Bound Nucleotides by the Measurement of Proton-Proton Transferred Nuclear Overhauser Enhancements

The glycosidic bond torsion angles and the conformations of the ribose of MgZCATP, Mg2+ADP and Mg2+AdoPP[NH]P (magnesium adenosine 5'[/?,pimido] 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 'H-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 x (04'-C1 '-NY-C4) for Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P bound to the low-affinity site of FITC-modified Ca2+ATPase are z 270°, x 260" and x 240" respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ATPase, x lies in the range 240 280".