Hydrodynamic and spectroscopic studies of substrate binding to human recombinant deoxycytidine kinase.
Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, plays a key role in the activation of therapeutic nucleoside analogues by their 5'-phosphorylation. The structure of human dCK is still not known and the current work was undertaken to determine its oligomeric and secondary structure. Biophysical studies were conducted with purified recombinant human dCK. The Mr determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 +/- 1,000, indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with ellipticity values of -11,900 +/- 300 and -12,500 +/- 300 deg x cm2 x dmol(-1), respectively, indicating the presence of approximately 40% alpha-helix and 50% beta-structure. Circular Dichroism studies in the aromatic and far-ultraviolet range and UV difference spectroscopy indicated that binding of substrates to dCK reduced its alpha-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence demonstrated that deoxycytidine (the phosphate acceptor) and ATP (the phosphate donor) bound to different sites on dCK and fluorescence quenching revealed bimodal binding of deoxycytidine and unimodal binding of ATP. Spectroscopic studies indicated that substrate binding induced conformational changes, with the result that dCK exhibited different affinities for various substrates. These results are consistent with a random bi-bi kinetic mechanism of phosphorylation of dCyd with either ATP or UTP.
Mani
Rajam S
RS
Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada. rajam.mani@cancerboard.ab.ca
Usova
Elena V
EV
Eriksson
Staffan
S
Cass
Carol E
CE
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000737:chemistry; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D008024:Ligands; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014461:Ultracentrifugation; D014544:Uridine Triphosphate / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D008024:Ligands; D011994:Recombinant Proteins; D003841:Deoxycytidine; D000255:Adenosine Triphosphate; D003842:Deoxycytidine Kinase; D014544:Uridine Triphosphate