Phosphorus-31 nuclear magnetic resonance studies of the conformation of an ATP analog at the active site of sodium-potassium ATPase from kidney medulla

Abstract

There are 2 sites for divalent metals at the active site of [sheep] kidney (Na+ + K+)-ATPase, one bound directly to the enzyme and one coordinated to the ATP substrate. The conformation of the metal-nucleotide complex was studied by using .beta.,.gamma.-bidentate Co(NH3)4ATP, a substitution-inert analog 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 .mu.M and Ki = 1.6 mM, respectively) are similar to the Km values for MnATP under the same conditions (2.88 .mu.M 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 3 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. Roles for both of the active site divalent cations are discussed.