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.

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.

Fluorescence energy transfer studies of human deoxycytidine kinase: role of cysteine 185 in the conformational changes that occur upon substrate binding.

Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the Förster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.