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.
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.
Schoner
W
W
Institute of Biochemistry and Endocrinology, Justus-Liebig-University Giessen, Germany.
Mertens
W
W
Helms
M
M
Fortes
G
G
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D009942:Organometallic Compounds / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013552:Swine; D062367:ortho-Aminobenzoates / Q000378:metabolism* / Q000494:pharmacology
D000345:Affinity Labels; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D062367:ortho-Aminobenzoates; C020511:tetraamminecobalt(III)ATP; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase