Binding of the Co(NH3)4 derivative of (2')3'-O-[N-methyl-anthraniloyl]-ATP to the E2ATP site of Na+/K+-transporting ATPase lowers the conformational flexibility of its E1ATP site.

Abstract

Na+/K+-activated ATP hydrolysis by the sodium pump is catalyzed by the interaction of high-affinity and low-affinity ATP-binding sites [Thoenges, D. & Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321]. To explore how binding of ATP to the low-affinity E2ATP site affects the conformational flexibility of the high-affinity E1ATP site due to interaction with Na+ or K+ ions, the E2ATP site was blocked with a fluorescent MgATP complex analog and fluorescence changes of the E1ATP site modified by FITC (fluorescein 5'-isothiocyanate) were studied. The fluorescent MgATP complex analog Co(NH3)4MANT-ATP [beta,gamma-bidentate complex of 2'(3')-O-(N-methylanthraniloyl)-ATP] inactivated Na+/K+-ATPase in a time-dependent and concentration-dependent process with a Kd of 0.17 mM and an inactivation rate constant, k, of 0.031 min(-1). ATP protected against the inactivation with a Kd of 0.43 mM. Consistent with a modification of the E2ATP binding site, Co(NH3)4MANT-ATP inactivated the K+-activated phosphatase activity in an enzyme whose E1ATP site had already been modified by FITC. Inactivation by Co(NH3)4MANT-ATP was due to tight binding which resulted in a loss of fluorescence. Tightly bound Co(NH3)4MANT-ATP could only be released by denaturation with SDS. Analysis of the conformational flexibility of the E1ATP site after labeling with FITC led to a K+-dependent quench of fluorescence which is reversed by Na+. This flexibility was lost upon the blockade of the E2ATP site by Co(NH3)4MANT-ATP.