1H nuclear magnetic resonance studies of the conformation of an ATP analogue at the active site of Na,K-ATPase from kidney medulla.
1H 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 previous 31P 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 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 bound Mn2+ lies above and in the plane of the adenine ring. The distances from 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Stewart
J M
JM
Department of Chemistry, University of Virginia, Charlottesville 22901.
Grisham
C M
CM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003035:Cobalt / Q000378:metabolism*; D006859:Hydrogen; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000494:pharmacology; D008968:Molecular Conformation; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D003035:Cobalt; D008345:Manganese; D006859:Hydrogen; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase