H-1-NMR STUDIES ON NUCLEOTIDE BINDING TO THE SARCOPLASMIC-RETICULUM CA-2+ ATPASE - DETERMINATION OF THE CONFORMATIONS OF BOUND NUCLEOTIDES BY THE MEASUREMENT OF PROTON-PROTON TRANSFERRED NUCLEAR OVERHAUSER ENHANCEMENTS

  • 1 January 1982
    • journal article
    • research article
    • Vol. 128  (1) , 113-117
Abstract
The glycosidic bond torsion angles and the conformations of the ribose of Mg2+ ATP, Mg2+ ADP and Mg2+ AdoPP[NH]P (magnesium adenosine 5''-[.beta.,.gamma.-imido]triphosphate) bound to Ca2+ ATPase, both native and modified with fluorescein isothiocyanate (FITC), in intact [rabbit] sarcoplasmic reticulum were 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 3 nucleotides bound to both the high-affinity (catalytic) site and the low-affinity site, 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 .chi. (04''-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 .apprxeq. 270.degree., .apprxeq. 260.degree. and .apprxeq. 240.degree., respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ ATPase, .chi. lies in the range 240-280.degree.