The effects of high salt concentrations on the regulation of the substrate specificity of human recombinant deoxycytidine kinase.
Deoxycytidine kinase (dCK) is a salvage pathway enzyme with broad substrate specificity that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. The kinetic behaviour of dCK is complex with saturation curves showing negative cooperativity. In this study, we have expressed and affinity purified recombinant dCK, using the pET 9d vector system with a histidine tag-sequence and a thrombin cleavage site fused to the N-terminus of the dCK coding sequence. The His-tagged protein showed essentially the same kinetic properties as the protease cleaved protein and the purified protein isolated from human spleen. However, addition of 0.2-0.4 M NaCl during the dCK reaction caused a stimulation of 2'-deoxycytidine (dCyd), and the antileukemic analog 2-chlorodeoxyadenosine (CldAdo) phosphorylation, but an inhibition of the 2'-deoxyguanosine (dGuo) phosphorylation, both with His-tagged and protease cleaved dCK. The negative cooperativity observed with dCyd was eliminated by the presence of 0.4 M NaCl so that the Hill coefficient changed from 0.6 to 1.4. In contrast, dGuo phosphorylation that initially followed Michaelis-Menten kinetics showed negative cooperativity after addition of 0.4 NaCl. The alterations in kinetic properties were not accompanied by any apparent changes in subunit structure as revealed by gel filtration. The major form of dCK eluted in the position corresponding to a dimer in the presence or absence of salt, but a minor fraction of dCK, eluting in the position of a tetramer, was diminished in the presence of salt. The mechanism for the effects of 0.4 M NaCl on dCK kinetic behaviour is not known but it is most likely due to alterations in the conformation of the active site of the enzyme. The effects described here also may explain some of the discrepancies reported in the literature on the substrate specificity of this complex enzyme.
Usova
E V
EV
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala.
Eriksson
S
S
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017338:Cladribine / Q000378:metabolism; D003001:Cloning, Molecular; D003841:Deoxycytidine / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003849:Deoxyguanosine / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology*; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D003841:Deoxycytidine; D012965:Sodium Chloride; D017338:Cladribine; D003842:Deoxycytidine Kinase; D003849:Deoxyguanosine