Fluorescence Studies of Substrate Binding to Human Recombinant Deoxycytidine Kinase

  title={Fluorescence Studies of Substrate Binding to Human Recombinant Deoxycytidine Kinase},
  author={Rajam S. Mani and Elena V. Usova and Staffan K Eriksson and Carol E. Cass},
  journal={Nucleosides, Nucleotides \& Nucleic Acids},
  pages={1343 - 1346}
  • R. Mani, E. Usova, C. Cass
  • Published 1 October 2004
  • Biology, Chemistry
  • Nucleosides, Nucleotides & Nucleic Acids
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… 
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Binding of substrates to human deoxycytidine kinase studied with ligand-dependent quenching of enzyme intrinsic fluorescence.
Fluorescence quenching was used to directly determine enzyme-ligand binding and revealed bimodal binding of dCTP, dCyd, dTTP, and dAdo and unimodalbinding of dUrd, and of d ado in the presence of 0.1 microM dCymru.
Kinetic analysis of human deoxycytidine kinase with the true phosphate donor uridine triphosphate.
The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phosphorylation by dCyd kinase, and indicate that the enzyme can follow a random bi-bi reaction sequence, but with UTP as the phosphate donor, the addition of nucleotide prior todCyd is strongly preferred.
Hydrodynamic and Spectroscopic Studies of Substrate Binding to Human Recombinant Deoxycytidine Kinase
Spectroscopic studies indicated that substrate binding induced conformational changes, with the result that dCK exhibited different affinities for various substrates.
A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: a model for nucleoside kinase action.
It is concluded that ATP and UTP induce a conformational change in the enzyme, thereby enabling efficient phosphoryl transfer.
Identification of residues involved in the substrate specificity of human and murine dCK.
Substrate Specificities, Expression and Primary Sequences of Deoxynucleoside Kinases; Implications for Chemotherapy
The biochemical properties of the purified enzymes and the sequences of their cDNA;s have been determined and TK2 and dGK show high homology to dCK and the herpes virus kinases but not to TK1.