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-Nemethy-Filmer model of two interacting ATP sites

A Koshland-Nemethy-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 E(1)ATP site (K-d = 0.7 mu M) and to the E(2)ATP site (K-d = 210 mu M), but 3'(2')-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E(1)ATP binding site (K-d = 200 mu M) than to the E(2)ATP binding site (K-d = 80 mu M). 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 mu M). 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 E(2)ATP 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 E(2)ATP site.