Tight binding of bulky fluorescent derivatives of adenosine to the low affinity E(2)ATP 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 adequate ly describes all types of interactions and kinetics of a number of ATP anal ogs that differ in their cooperativity of the high and low affinity ATP bin ding sites of the enzyme. 2',3'-O(2,4,6-trinitrophenyl)ATP binds in a negat ive 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 c ooperative 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 a ffinities to both ATP binding sites (K-d = 10 mu M). in a research for the structural parameters determining ATP site specificity and cooperativity, w e became aware that structural flexibility of ribose is necessary for catal ysis, 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 af finity to the E(2)ATP site and inhibit Na+/K+-ATPase activity. Evidently, a n increased number of interactions of such a bulky adenosine with the enzym e protein tightens binding to the E(2)ATP site.