Identification of residues involved in the substrate specificity of human and murine dCK.
Deoxycytidine kinase (dCK) is a salvage pathway enzyme that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. Earlier studies showed that there are differences in kinetic properties between human and murine dCK, which may explain differences in toxic effects of nucleoside analogs. To determine if certain substitutions in amino acid sequences between human and mouse dCK give these differences in substrate specificity the 14 mutants and hybrid forms of human dCK were studied. All variants were characterised with dCyd, dAdo and dGuo as phosphate acceptors and ATP and UTP as phosphate donor. The relative activities with dCyd, dAdo and dGuo were about 70, 20, 30%, respectively, with UTP as compared to ATP for human dCK and 40, 60, 70% for mouse dCK. Among all tested mutants only the triple combination of substitutions Q179R-T184K-H187N (RKN) had a kinetic behaviour very similar to mouse dCK. The kinetic patterns with several important nucleoside analogs, such as AraC, CdA, ddC and AraG have also been studied. Results demonstrated 50-70% low relative capacities of the recombinant mouse and triple mutant RKN to phosphorylate this nucleoside analogs compare with human dCK. A model for dCK was used to try to explain the functional role of these amino acid substitutions. According to this model the triple mutant RKN have altered amino acids in a region necessary for conformational changes during catalyses. This may affects the substrate selectivity both for the nucleosides and the phosphate donors.
Usova
Elena V
EV
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Box 575, S-751 23, Uppsala, Sweden. elena.usova@vmk.slu.se
Eriksson
Staffan
S
D000595:Amino Acid Sequence; D000818:Animals; D003842:Deoxycytidine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity
D009705:Nucleosides; D011994:Recombinant Proteins; D003842:Deoxycytidine Kinase