¹H Nuclear Magnetic Resonance Studies of the Conformation of an ATP Analogue at the Active Site of Na,K-ATPase from Kidney Medulla

lh nuclear magnetic relaxation measurements 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., Klevickis, C., & Grisham, C. M. (1982) Biochemistry 21, 4083] and that Mn²⁺bound to a single, high-affinity site on the ATPase can be an effective paramagnetic probe for nuclear relaxation studies of the Na,K-ATPase [O'Connor, S. E., & Grisham, C. M. (1979) Biochemistry 18, 2315]. From the paramagnetic effect of Mn²⁺bound to the ATPase on the longitudinal relaxation rates of the protons of Co(NH₃)₄ATP at the substrate site (at 300 and 361 MHz), Mn-H distances to seven protons on the bound nucleotide were determined. Taken together with previous³¹P nuclear relaxation data, these measurements are consistent with a single nucleotide conformation at the active site. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. The glycosidic torsion angle is 35°, and the conformation of the ribose ring is slightly N-type (C_2'-exo, C_3'-endo). The δ and ϒ torsional angles in this conformation are 100° and 178°, respectively. The bound Mn²⁺lies above and in the plane of the adenine ring. The distances from Mn²⁺to N₆and N₇are 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.