Study of the substrate-binding properties of bovine liver adenosine kinase and inhibition by fluorescent nucleoside analogues.

Adenosine kinase (AK) catalyzes the phosphorylation of adenosine to AMP with ATP as phosphate donor. Intrinsic fluorescence of bovine liver AK was shown previously to be a sensitive probe to quantify the binding of substrates to the enzyme [Elaloui, A., Divita, G., Maury, G., Imbach, J.-L. & Goody, R. S. (1994) Eur. J Biochem. 221, 839-846]. AK contains two catalytic, sites: a high-affinity site, which binds adenosine and AMP selectively; and a site for ATP and ADP. In the present work, these two sites were characterized by combining the quenching of protein fluorescence induced by the binding of the ligands and the fluorescence enhancement observed upon binding of the N-methylanthraniloyl-derivated nucleotides or adenosine. A new fluorescent analog of adenosine, 5'-N-methylanthraniloyl-adenosine, was synthesized and shown to bind selectively to the high-affinity adenosine-binding site with an affinity similar to that of adenosine (Kd 1 microM). In contrast, 2'(3')-N-methylanthraniloyl derivatives of ATP, adenosine (5')tetraphospho(5')adenosine (Ap4A), and adenosine (5')pentaphospho(5')adenosine (Ap5A), bind to the enzyme at the ATP site. Methylantraniloyl derivatives of ATP and adenosine were used as tools for selective characterization of a series of adenosine analogues. The bisubstrate inhibitors Ap4A and Ap5A bind to the ATP site with high affinity and apparently not to the adenosine site, thus acting more as ATP analogues than true bisubstrate ligands. The binding properties of a series of adenosine analogues were strongly dependent on the structural modifications on adenosine. The analogues modified at positions 2' or 3' show similar affinities for AK as that of adenosine, whereas adenosine analogues modified at the base present a relatively low affinity for the enzyme.