Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase

doi:10.1016/0006-2952(95)00129-N

Biochemical Pharmacology

Volume 50, Issue 2, 17 July 1995, Pages 163-168

https://doi.org/10.1016/0006-2952(95)00129-N Get rights and content

Abstract

Human and mouse deoxycytidine kinase (dCK) (EC 2.7.1.74) were cloned and expressed in Escherichia coli. Michaelis-Menten kinetics were determined for the purified enzymes with 2′-deoxycytidine (dCyd), 2′-deoxyadenosine (dAdo), 2-chloro-2′-deoxyadenosine (CdA), 2′,3′-dideoxycytidine (ddCyd) and 9-β-d-arabinofuranosylguanine (araG) as substrates and ATP and UTP as phosphate donors. Both human and mouse dCK showed highest affinity to dCyd with K_m values of 0.05–0.2 μM. The anti-leukaemic compound CdA was the superior substrate of the nucleoside analogues tested. Both enzymes were able to efficiently utilize ATP and UTP as phosphate donors. However, the use of UTP instead of ATP as phosphate donor decreased K_m values for all substrates investigated. The kinetic properties of mouse and human dCK differed in that the human enzyme showed higher affinity for the substrates dAdo, CdA, ddCyd and araG. The human enzyme also showed higher affinity for ATP and UTP. The ability to phosphorylate dCyd was, however, similar for both human and mouse dCK. At physiological concentration of the feedback inhibitor dCTP, mouse dCK showed lower activity than human dCK for all substrates investigated.

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It should be noted that, in these experiments, 2CdA was used at concentrations significantly higher than those measured in CSF during clinical trials (0.019–0.025 µM) [62]. However, DCK activity is significantly lower in mice than in humans [63–64], which suggests differences in the susceptibility of a similar cell type between humans and mice. Furthermore, different concentrations of 2CdA influence different aspects of microglial function [47].
Cladribine (2CdA) is a synthetic chlorinated purine nucleoside analogue which acts as a pro-drug requiring intracellular phosphorylation to be activated. It is biologically active in selected cell types, which results in a reduction of circulating T and B lymphocytes implicated in multiple sclerosis (MS) pathogenesis. In addition, 2CdA shows good central nervous system (CNS) penetration and can therefore exert its action on microglia and astrocytes. Therefore, we studied the effects of 2CdA on microglial cells and astrocytes, both emerging as potential targets for MS therapy. Other than its effects on the peripheral immune system, 2CdA induced the apoptosis of microglial cells, inhibited their proliferation and reduced the production of cytokines, particularly pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. These represent additional mechanisms of 2CdA that may contribute to limiting inflammatory pathways. By contrast, astrocytes showed resistance to the action of 2CdA, which may be explained by differences in its intracellular phosphorylation. Insights into the mechanism of action of and resistance to 2CdA in CNS-resident cells may prove crucial for its optimal use.
Facile method for determination of deoxycytidine kinase activity in biological milieus
2014, Journal of Food and Drug Analysis
Citation Excerpt :
Deoxycytidine kinase (dCK) is an enzyme expressed in eukaryotic cells [1,2] that catalyzes phosphate transfer between adenosine triphosphate (ATP) and 2′-deoxynucleosides.
A new analytical method for determining deoxycytidine kinase (dCK) activity in biological milieus using luminescence is reported here. This method, based on utilizing adenosine triphosphate (ATP) as the sole phosphate donor in the kinase reaction and monitoring ATP consumption via a luciferase-based chemiluminescence reaction, is capable of detecting dCK activity without the use of specific substrates or radioisotope techniques. Comparing with the reverse-phase high-performance liquid chromatography method, this new method is suggested to be efficient and sensitive. Further, application of the proposed method for profiling dCK activity in cultured cancer cells revealed that a cervix cell line exhibited the highest dCK activity to gemcitabine metabolism.
Identification of in vivo phosphorylation sites on human deoxycytidine kinase: Role of Ser-74 in the control of enzyme activity
2006, Journal of Biological Chemistry
Citation Excerpt :
Size exclusion chromatography (Sephacryl S-200) gave a single peak of dCK activity eluting with a molecular mass of 68 kDa, indicating that the active form of recombinant dCK is a dimer (result not shown), as reported for the native enzyme (30). The Kmvalue of purified recombinant (His)6dCK for deoxycytidine, with 5 mm ATP as co-substrate, was 0.16 ± 0.01 μm (n = 3), in agreement with values reported previously for dCK expressed in bacteria (31). Extracts of HEK 293T cells were incubated with λ-protein phosphatase, which led to a marked (∼ 85%) decrease in dCK activity (Fig. 1A), as observed previously for the native enzyme from normal (21) or leukemic human lymphocytes (24).
Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [³²P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of ³²P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.
Detection of an alternatively spliced form of deoxycytidine kinase mRNA in the 2′-2′-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000
2004, Biochemical Pharmacology
Citation Excerpt :
Deoxycytidine kinase (dCK, EC 2.7.1.74) is a salvage pathway enzyme that catalyses the phosphorylation of deoxycytidine (dC) into dCMP, using ATP or UTP as phosphate donors [1–3].
Gemcitabine (2′-2′-difluorodeoxycytidine (dFdC)) is a deoxycytidine analogue that is effective against solid tumors, including lung cancer and ovarian cancer. dFdC requires the phosphorylation by deoxycytidine kinase (dCK) as a primary step in its activation. Deficiency of dCK is associated with resistance against this compound both in vitro in cancer cell lines and in clinical practice in acute myeloid leukemia and solid tumors. The human ovarian cancer cell line AG6000 is 100,000-fold resistant against dFdC compared to its parent cell line A2780. This cell line proved to be dCK deficient in enzyme activity assays and by Western blot analysis, but by RT-PCR, a normal and a truncated dCK mRNA was found. Sequencing revealed that exon 3 was deleted from the dCK cDNA, resulting in a 74-aa-long open-reading frame due to the generation of a premature stop codon. No gross genomic alteration was observed at the dCK locus, suggesting the involvement of post-transcription mechanisms. Transient transfection experiments indicated that the truncated dCK transcripts are not translated to protein. To study the functional role of the truncated dCK transcripts, both A2780 cells and AG6000 cells were stably transfected with human and rat dCK. The results indicated that over-expression of full-length dCK genes in AG6000 failed to completely reverse the sensitivity to dFdC or other drugs.
Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by human deoxyribonucleoside kinases and ribonucleoside kinases
2003, Pharmacology and Therapeutics
Structural analogues of nucleosides, nucleoside analogues (NA), are used in the treatment of cancer and viral infections. Antiviral NAs inhibit replication of the viral genome, whereas anticancer NAs inhibit cellular DNA replication and repair. NAs are inactive prodrugs that are dependent on intracellular phosphorylation to their pharmacologically active triphosphate form. The deoxyribonucleoside kinases (dNK) and ribonucleoside kinases (rNK) catalyze the first phosphorylation step, converting deoxyribonucleosides and ribonucleosides to their corresponding monophosphate form. The dNKs have been studied intensively, whereas the rNKs have not been as thoroughly investigated. This overview is focused on the substrate specificity, tissue distribution, and subcellular location of the mammalian dNKs and rNKs and their role in the activation of NAs.
Identification of residues involved in the substrate specificity of human and murine dCK
2002, Biochemical Pharmacology
Citation Excerpt :
Although the amino acid sequences are 88% identical between mouse and human dCK the enzymes have different kinetic behaviour. Both enzymes had a broad substrate specificity phosphorylating many pyrimidine and purine analogs, but the mouse enzyme was less efficient in phosphorylating dAdo, dGuo, ddC as compared to human dCK [15,16]. ATP and UTP, as well as many other nucleoside triphosphates can serve as phosphate donors for 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.

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Biochemical Pharmacology

Abstract

References (18)

Deoxynucleoside phosphorylating enzymes in monkey and human tissues show great similarities, while mouse deoxycytidine kinase has a different substrate specificity

Biochem Pharmacol

The in vitro and in vivo anti-retrovirus activity, and intracellular metabolism of 3′-azido-2′,3′-dideoxythymidine and 2′,3′-dideoxycytidine are highly dependent on the cell species

Biochem Pharmacol

Cloning and expression of mouse deoxycytidine kinase; Pure recombinant mouse and human enzymes show differences in substrate specificity

J Biol Chem

Rapid determination of nucleoside kinase and nucleotidase activities with tritium-labeled substrates

Anal Biochem

Deoxycytidine kinase. III. Kinetics and allosteric regulation of the calf thymus enzyme

J Biol Chem

Substrate specificity of human deoxycytidine kinase toward antiviral 2′,3′-dideoxynucleoside analogs

Biochem Pharmacol

Regulation of purine deoxynucleoside phosphorylation by deoxycytidine kinase from human leukemic blast cells

Biochem Pharmacol

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

J Biol Chem

Initial studies on the cellular pharmacology of 2′,3′-dideoxycytidine, an inhibitor of HTLV-III infectivity

Biochem Pharmacol

Cited by (28)

2-Chlorodeoxyadenosine (Cladribine) preferentially inhibits the biological activity of microglial cells

Facile method for determination of deoxycytidine kinase activity in biological milieus

Identification of in vivo phosphorylation sites on human deoxycytidine kinase: Role of Ser-74 in the control of enzyme activity

Detection of an alternatively spliced form of deoxycytidine kinase mRNA in the 2′-2′-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000

Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by human deoxyribonucleoside kinases and ribonucleoside kinases

Identification of residues involved in the substrate specificity of human and murine dCK

Research paperDifferences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase

Abstract

Biochem Pharmacol

Biochem Pharmacol

J Biol Chem

Anal Biochem

J Biol Chem

Biochem Pharmacol

Biochem Pharmacol

J Biol Chem

Biochem Pharmacol

Research paper
Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase