Study of human deoxycytidine kinase binding properties using intrinsic fluorescence or new fluorescent ligands

• Published in: European journal of medicinal chemistry 1999-05, Vol.34 (5), p.423-431
• Main Authors: Shafiee, Manijeh, Gosselin, Gilles, Imbach, Jean-Louis, Divita, Gilles, Eriksson, Staffan, Maury, Georges
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; deoxycytidine kinase; enantioselectivity; Enzymes and enzyme inhibitors; fluorescent cytidine derivatives; Fundamental and applied biological sciences. Psychology; Transferases
• ISSN: 0223-5234; 1768-3254
• DOI: 10.1016/S0223-5234(99)80092-0

Aseries of D- and L-enantiomers of cytidine or adenosine analogues and fluorescent N-methylanthraniloyl (MeNHBz) cytidine derivatives regiospecifically synthesized from cytidine or deoxycytidine were used to quantify the enantioselectivity of human deoxycytidine kinase (dCK) and to characterize its binding states. Both D- and L-enantiomers of these compounds induced significant bimodal quenchings of the intrinsic fluorescence of the enzyme. The ratios of dissociation constants Kd D/Kd L for the high affinity binding of non fluorescent cytidine derivatives were remarkably similar. β-D- and β-L-ATP gave monophasic titration curves and the enzyme displayed a preference for the natural enantiomer. This demonstrates the lack of enantioselectivity of dCK in the substrate binding steps of its mechanism. The results of other fluorescence experiments with MeNHBz-cytidine derivatives were consistent with an enzyme mechanism in which nucleotide binding precedes nucleoside binding.