Human deoxycytidine kinase as a deoxyribonucleoside phosphorylase

Human deoxycytidine kinase (dCK) is a key enzyme in the 5'-phosphorylation of purine and pyrimidine deoxynucleosides with deoxycytidine as the most efficient substrate. The ability of dCK to degrade 2'-deoxyribonucleosides to free nucleobases and 2-deoxy-alpha-d-ribofuranose-1-phosphate was demonstrated by 1H-31P correlation spectroscopy and by isotope enzyme kinetic methods. The reaction depended on inorganic phosphate, and dCK showed maximum cleavage activity between pH 7 and pH 8. In this pH range, [HPO4(2-)] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the Vmax of the phosphorylytic reaction compared to the kinase reaction was about 2-10%. The formation of free nucleobases occurred only with reduced dCK, because the reaction was highly dependent on the presence of reducing agents such as dithiotreitol. Thus, recombinant dCK can act as a phosphorylase, similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is important for the design of in vitro experiments with dCK, such as crystallization and NMR spectroscopy.     

Human deoxycytidine kinase: kinetic mechanism and end product regulation

The kinetic properties of the monomeric deoxycytidine kinase (EC 2.7.1.74) from leukemic human T-lymphoblasts have been investigated. The results of steady-state initial-rate kinetic analysis and product inhibition studies at pH 7.5 and 37 degrees C indicate that substrate binding follows an ordered sequential pathway, with the magnesium salt of ATP being the first substrate to bind and dCMP the last product to dissociate. At subsaturating substrate concentrations, dCMP produced competitive inhibition against ATP, while against varied deoxycytidine concentrations dCMP exhibited mixed-type inhibition. ADP produced noncompetitive inhibition against either substrate. The limiting Km values for deoxycytidine and MgATP were 0.94 and 30 microM, respectively. The end product inhibitor dCTP exhibited competitive inhibition against varied ATP concentration, with a dissociation constant estimated to be 0.7 microM when extrapolated to zero ATP concentration. dCTP was purely noncompetitive against varied deoxycytidine concentration. On the basis of these kinetic results, and on the strong and specific inhibition by dCTP, it is proposed that this end product functions as a multisubstrate analogue, with its triphosphate group binding to the phosphate donor site of the enzyme and its deoxycytidine moiety overlapping and binding to the deoxynucleoside site in a highly specific manner.     

Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity

Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.     

Deoxycytidine kinase from human leukemic spleen: preparation and characteristics of homogeneous enzyme

Deoxycytidine kinase from human leukemic spleen has been purified 6000-fold to apparent homogeneity with an overall yield of 10%. The purification was achieved by using DEAE chromatography, hydroxylapatite chromatography, and affinity chromatography on dTTP-Sepharose. Only one form of deoxycytidine kinase activity was found during all the chromatographic procedures. The subunit molecular mass, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, was 30 kilodaltons. The pure enzyme phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, demonstrating for the first time that the same enzyme molecule has the capacity to use these three nucleosides as substrates. The apparent molecular weight of the active enzyme, determined by gel filtration and glycerol gradient centrifugation, was 60,000. Thus, the active form of human deoxycytidine kinase is a dimer. The kinetic behavior of pure human deoxycytidine kinase was studied in detail with regard to four different phosphate acceptors and two different phosphate donors. The apparent Km values were 1, 20, 150, and 120 microM for deoxycytidine, arabinosylcytosine, deoxyguanosine, and deoxyadenosine, respectively. The Vmax values were 5-fold higher for the purine nucleosides as compared to the pyrimidine substrates. We observe competitive inhibition of the phosphorylation of one substrate by the presence of either of the three other substrates, but the apparent Ki values differed greatly from the corresponding Km values, suggesting the existence of allosteric effects. The double-reciprocal plots for ATP-MgCl2 as phosphate donor were convex, indicating negative cooperative effects. In contrast, plots with varying dTTP-MgCl2 concentration as phosphate donor were linear with an apparent Km of 2 microM. The enzyme activity was strongly inhibited by dCTP, in a noncompetitive way with deoxycytidine and in a competitive way with ATP-MgCl2.     

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.     

Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological function

Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis. The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography. The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis. The molecular weight is determined by gel filtration to be 47,000. Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme. The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine. The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM. The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor. GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration. dATP and dCTP are inhibitors when GTP is the phosphate donor. They may both act as phosphate donors themselves. A divalent metal ion is required, Mg2+ giving the highest activity. A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine.     

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.     

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5′ exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R² values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.     

Structural basis for the preference of UTP over ATP in human deoxycytidine kinase: illuminating the role of main-chain reorganization

Human deoxycytidine kinase (dCK) uses nucleoside triphosphates to phosphorylate several clinically important prodrugs in addition to its natural substrates. Although UTP is the preferred phosphoryl donor for this reaction, our previous studies reported dCK structures solely containing ADP in the phosphoryl donor binding site. To determine the molecular basis of the kinetically observed phosphoryl donor preference, we solved crystal structures of a dCK variant lacking a flexible insert (residues 65-79) but having similar catalytic properties as wild type, in complex with deoxycytidine (dC) and UDP, and in the presence of dC but the absence of UDP or ADP. These structures reveal major changes in the donor base binding loop (residues 240-247) between the UDP-bound and ADP-bound forms, involving significant main-chain rearrangement. This loop is disordered in the dCK-dC structure, which lacks a ligand at the phosphoryl donor site. In comparison with the ADP-bound form, in the presence of UDP this loop is shifted inward to make closer contact to the smaller uracil base. These structures illuminate the phosphoryl donor binding and preference mechanisms of dCK.     

Low enantioselectivities of human deoxycytidine kinase and human deoxyguanosine kinase with respect to 2'-deoxyadenosine, 2'-deoxyguanosine and their analogs

The antiviral activity of L-nucleoside analogs depends in part on the enantioselectivity of nucleoside kinases which catalyse their monophosphorylation. The substrate properties of human recombinant deoxycytidine kinase (dCK) and human recombinant deoxyguanosine kinase (dGK) with respect to L-adenosine and L-guanosine analogs, in the presence of saturating amounts of ATP and relatively high concentrations of substrates, demonstrated a marked lack of enantioselectivity of both these enzymes. Human dCK catalysed the phosphorylation of D- and L-enantiomers of beta-dA, beta-araA, and beta-dG with enantioselectivities favoring the unnatural enantiomer for the adenosine derivatives and the natural enantiomer for 2'-deoxyguanosine. No other tested L-adenosine or L-guanosine analog was a substrate of dCK. Similarly, D- and L-enantiomers of beta-dA, beta-araA, and beta-dG were substrates of human dGK but with different enantioselectivities compared to dCK, especially concerning beta-dA. The present results indicate that human dCK and dGK have similar properties including substrate properties, relaxed enantioselectivities, and possibly catalytic cycles.     

Fluorescence studies of substrate binding to human recombinant deoxycytidine kinase

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 dCK can assume different conformational states with different substrates and may account for the observed differences in their specificity. dCK bound the substrates more tightly in the presence of phosphate donors and UTP is the preferred phosphate donor. Among the substrates tested, the antitumour drugs gemcitabine and cladribine were bound very tightly by dCK, yielding Kd values of 0.75 and 0.8 microM, respectively, in the presence of UTP.     

Inorganic tripolyphosphate (PPP(i)) as a phosphate donor for human deoxyribonucleoside kinases

Inorganic tripolyphosphate (PPP(i)) and pyrophosphate (PP(i)) were examined as potential phosphate donors for human deoxynucleoside kinase (dCK), deoxyguanosine kinase (dGK), cytosolic thymidine kinase (TK1), mitochondrial TK2, and the deoxynucleoside kinase (dNK) from Drosophila melanogaster. PPP(i) proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not dAdo, as acceptor substrate, illustrating also the dependence of donor properties on acceptor. Products of phosphorylation were shown to be 5(')-phosphates. In striking contrast to ATP, the phosphorylation reaction follows strict Michaelis-Menten kinetics, with K(m) values of 74 and 92 microM for dCK and dGK, respectively, and V(max) values 40-50% that for ATP. With the other three enzymes, as well as for dCK with dAdo as acceptor, no, or only low levels (相似文献   

Deoxycytidine Kinase Augments ATM-Mediated DNA Repair and Contributes to Radiation Resistance

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [¹⁸F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.     

Potential of ribozymes against deoxycytidine kinase to confer drug resistance to cytosine nucleoside analogs

Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs.     

Activation of deoxycytidine kinase by deoxyadenosine: implications in deoxyadenosine-mediated cytotoxicity

The inborn deficiency of adenosine deaminase is characterised by accumulation of excess amounts of cytotoxic deoxyadenine nucleotides in lymphocytes. Formation of dATP requires phosphorylation of deoxyadenosine by deoxycytidine kinase (dCK), the main nucleoside salvage enzyme in lymphoid cells. Activation of dCK by a number of genotoxic agents including 2-chlorodeoxyadenosine, a deamination-resistant deoxyadenosine analogue, was found previously. Here, we show that deoxyadenosine itself is also a potent activator of dCK if its deamination was prevented by the adenosine deaminase inhibitor deoxycoformycin. In contrast, deoxycytidine was found to prevent stimulation of dCK by various drugs. The activated form of dCK was more resistant to tryptic digestion, indicating that dCK undergoes a substrate-independent conformational change upon activation. Elevated dCK activities were accompanied by decreased pyrimidine nucleotide levels whereas cytotoxic dATP pools were selectively enhanced. dCK activity was found to be downregulated by growth factor and MAP kinase signalling, providing a potential tool to slow the rate of dATP accumulation in adenosine deaminase deficiency.     

Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues.

Human cells salvage pyrimidine deoxyribonucleosides via 5'-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3'-azido-2',3'-dideoxythymidine (AZT) as substrate for TK1, 1-beta-D-arabinofuranosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.     

Thymidine and thymidylate kinases from the scallop <Emphasis Type="Italic">Mizuhopecten yessoensis</Emphasis> gonads

Thymidine and thymidylate kinases were isolated from the gonads of scallop Mizuhopecten yessoensis. The enzymes were purified 537-and 100-fold, respectively, and were free of phosphatase and ATPase impurities. Ions of bivalent metals and ATP were necessary for both the nucleoside and nucleotide kinase activities; the pH optimum fall into the range of 7.5–8.5. KCl and NaCl at a concentration of up to 100 mM had no inhibiting effect on the activities of these scallop enzymes. Thymidine kinase catalyzed thymidine, and, at a lower rate, deoxycytidine phosphorylations did not utilize ribo-and deoxyribonucleosides, as well as pyrimidine ribonucleosides, as a phosphate acceptor. Thymidylate kinase phosphorylated TMP and dCMP with an efficiency of about 30%. In addition to ATP, these enzymes can also utilize with different efficiencies dATP, dGTP, GTP, UTP, and CTP as a donor of phosphate groups. Thymidine kinase activity was inhibited by TMP, TTP, and dCTP.     

The purification and characterization of deoxycytidine kinase from calf thymus

A facile and fast approach for the purification of deoxycytidine kinase (dCK) from calf thymus was developed using a fast performance liquid chromatography system. A 73-fold enrichment of the enzyme was noted compared to unfractionated dCK. Characterization studies demonstrated that dCK had a molecular mass of 31 kDa using SDS–PAGE, an optimum pH of 7.0 and the enzyme maintained stability between 30 and 40°C. The rapid preparation of dCK demonstrated here will be valuable in the synthesis of nucleotide analogs.     

Thymidine and thymidylate kinases from the scallop Mizuhopecten yessoensis gonads

Thymidine and thymidylate kinases were isolated from the gonads of scallop Mizuhopecten yessoensis. The enzymes were purified 537- and 100-fold, respectively, and were free of phosphatase and ATPase impurities. Ions of bivalent metals and ATP were necessary for both the nucleoside and nucleotide kinase activities; the pH optimum fall into the range of 7.5-8.5. KCl and NaCl at a concentration of up to 100 mM had no inhibiting effect on the activities of these scallop enzymes. Thymidine kinase catalyzed thymidine, and, at a lower rate, deoxycytidine phosphorylations did not utilize ribo- and deoxyribonucleosides, as well as pyrimidine ribonucleosides, as a phosphate acceptor. Thymidylate kinase phosphorylated TMP and dCMP with an efficiency of about 30%. In addition to ATP, these enzymes can also utilize with different efficiencies dATP, dGTP, GTP, UTP, and CTP as a donor of phosphate groups. Thymidine kinase activity was inhibited by TMP, TTP, and dCTP.     

Human cytomegalovirus induces a cellular deoxyguanosine kinase, also interacting with Acyclovir

Abstract A cytosol deoxyguanosine kinase (dGK) is induced in either growing or human cytomegalovirus (HCMV, AD169)-infected human fibroblasts (HEF). Data obtained from polyacrylamide gel electrophoresis, heat inactivation and phosphorylation kinetic experiments proved that these dGKs are identical, but completely differ from HCMV-induced thymidine kinase (TK) or deoxycytidine kinase (dCK). In contrast to TK or dCK, only dGK interacts with Acyclovir ( K _i = 590 μ M). It is suggested that dGK is an important enzyme determining the antiviral activity of Acyclovir.