title: Nuclear Overhauser effect studies of the conformation of Co(NH3)4ATP bound to kidney Na,K-ATPase.
abstract: Transferred nuclear Overhauser effect measurements (in the two-dimensional mode) have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase [Klevickis, C., & Grisham, C.M. (1982) Biochemistry 21, 6979. Gantzer, M.L., et al. (1982) Biochemistry 21, 4083]. Nine unique proton-proton distances on ATPase-bound Co(NH3)4ATP were determined from the initial build-up rates of the cross-peaks of the 2D-TRNOE data sets. These distances, taken together with previous 31P and 1H relaxation measurements with paramagnetic probes, are consistent with a single nucleotide conformation at the active site. The bound Co(NH3)4ATP) adopts an anti conformation, with a glycosidic torsion angle of 35 degrees, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. Mn2+ bound to a single, high-affinity site on the ATPase lies above and in the plane of the adenine ring. The distances from enzyme-bound Mn2+ to N6 and N7 are too large for first coordination sphere complexes, but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville 22901.
authors.forename: J M
authors.initials: JM
authors.lastname: Stewart
authors.forename: P L
authors.initials: PL
authors.lastname: Jørgensen
authors.forename: C M
authors.initials: CM
authors.lastname: Grisham
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D007668:Kidney / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009942:Organometallic Compounds; D011522:Protons; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D009942:Organometallic Compounds; D011522:Protons; C020511:tetraamminecobalt(III)ATP; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase