Phosphorus-31 nuclear magnetic resonance studies of the conformation of an adenosine 5'-triphosphate analogue at the active site of (Na+ + K+)-ATPase from kidney medulla.
It has previously been shown that there are two sites for divalent metals at the active site of kidney (Na+ + K+)-ATPase, one bound directly to the enzyme and one coordinated to the ATP substrate [Grisham, C. (1981) J. Inorg. Biochem. 14, 45; O'Connor, S., & Grisham, C. (1980) FEBS Lett. 118, 303]. The conformation of the metal-nucleotide complex has been studied by using beta, gamma-bidentate Co-(NH3)4ATP, a substitution-inert analogue of MgATP. Kinetic studies show that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the (Na+ + K+)-ATPase. The Ki values under both high- and low-affinity conditions (Ki = 10 microM and Ki = 1.6 mM, respectively) are similar to the Km values for MnATP under the same conditions (2.88 microM and 0.902 mM). From the paramagnetic effect of Mn2+ bound to the ATPase on the longitudinal relaxation rates of the phosphorus nuclei of Co(NH3)4ATP at the substrate site (at 40.5 and 145.75 MHz), Mn-P distances to all three phosphates are determined. The distances are consistent with the formation of a second sphere coordination complex on the enzyme between Mn2+ and the phosphates of Co(NH3)4ATP. In this respect, kidney (Na+ + K+)-ATPase appears to be similar to pyruvate kinase [Sloan, D., & Mildvan, A. (1976) J. Biol. Chem. 251, 2412] and phosphoribosylpyrophosphate synthetase [Granot, J., Gibson, K., Switzer, R., & Mildvan, A. (1980) J. Biol. Chem. 255, 10931]. Roles for both of the active site divalent cations are discussed.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008958:Models, Molecular; D009942:Organometallic Compounds; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D009942:Organometallic Compounds; C020511:tetraamminecobalt(III)ATP; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase