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J Biol Chem, Vol. 274, Issue 4, 1971-1978, January 22, 1999

Tight Binding of Bulky Fluorescent Derivatives of Adenosine to the Low Affinity E₂ATP Site Leads to Inhibition of Na⁺/K⁺-ATPase
ANALYSIS OF STRUCTURAL REQUIREMENTS OF FLUORESCENT ATP DERIVATIVES WITH A KOSHLAND-NÉMETHY-FILMER MODEL OF TWO INTERACTING ATP SITES

Detlef Thoenges, Evzen Amler^§, Thomas Eckert^¶, and Wilhelm Schoner

From the  Institute of Biochemistry and Endocrinology and ^¶ Institute of Organic Chemistry, Justus-Liebig-University Giessen, D-35392 Giessen, Germany and ^§ Institute of Physiology, Czech Academy of Sciences, Videnska 1083, Cz-142 20 Prague 4, Czech Republic

A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na⁺/K⁺-ATPase (EC 3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321). The present work demonstrates that this model adequately describes all types of interactions and kinetics of a number of ATP analogs that differ in their cooperativity of the high and low affinity ATP binding sites of the enzyme. 2',3'-O(2,4,6-trinitrophenyl)ATP binds in a negative cooperative way to the E₁ATP site (K_d = 0.7 µM) and to the E₂ATP site (K_d = 210 µM), but 3'(2')-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E₁ATP binding site (K_d = 200 µM) than to the E₂ATP binding site (K_d = 80 µM). 3'(2')-O(5-Fluor-2,4-dinitrophenyl)-ATP, however, binds in a noncooperative way, with equal affinities to both ATP binding sites (K_d = 10 µM). In a research for the structural parameters determining ATP site specificity and cooperativity, we became aware that structural flexibility of ribose is necessary for catalysis. Moreover, puckering of the ring atoms in the ribose is essential for the interaction between ATP sites in Na⁺/K⁺-ATPase. A number of derivatives of 2'(3')-O-adenosine with bulky fluorescent substitutes bind with high affinity to the E₂ATP site and inhibit Na⁺/K⁺-ATPase activity. Evidently, an increased number of interactions of such a bulky adenosine with the enzyme protein tightens binding to the E₂ATP site.

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