Fluorescence studies of substrate binding to human recombinant deoxycytidine kinase.
Deoxycytidine kinase (dCK), is responsible for the phosphorylation of deoxynucleosides to the corresponding monophosphates using ATP or UTP as phosphate donors. Steady-state intrinsic fluorescence measurements were used to study interaction of dCK with substrates in the absence and presence of phosphate donors. Enzyme fluorescence quenching by its substrates exhibited unimodal quenching when excited at 295 nm. Binding of substrates induced conformational changes in the protein, suggesting that dCK can assume different conformational states with different substrates and may account for the observed differences in their specificity. dCK bound the substrates more tightly in the presence of phosphate donors and UTP is the preferred phosphate donor. Among the substrates tested, the antitumour drugs gemcitabine and cladribine were bound very tightly by dCK, yielding Kd values of 0.75 and 0.8 microM, respectively, in the presence of UTP.
Mani
R S
RS
Department of Oncology, University of Alberta, Edmonton, Alberta, Canada. rajam.mani@cancerboard.ab.ca
Usova
E V
EV
Eriksson
S
S
Cass
C E
CE
D000255:Adenosine Triphosphate / Q000737:chemistry; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence; D009369:Neoplasms / Q000188:drug therapy; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014544:Uridine Triphosphate / Q000737:chemistry; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D008024:Ligands; D010710:Phosphates; D011994:Recombinant Proteins; D003841:Deoxycytidine; D000255:Adenosine Triphosphate; D003842:Deoxycytidine Kinase; D014544:Uridine Triphosphate; D000093542:Gemcitabine