Human deoxycytidine kinase 2

Details Human deoxycytidine kinase 2 Human deoxycytidine kinase 2

1997-10-16

2000-05-16

Wax, Robert A.

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Transferases

435194, A61K 3845, C12N 912

Patent

active

06063376&

DESCRIPTION:

BRIEF SUMMARY
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is Human Deoxycytidine Kinase 2, sometimes hereinafter referred to as "hdCK2". The invention also relates to inhibiting the action of such polypeptides.
Human deoxycytidine kinase 2 is responsible for the phosphorylation of several deoxyribonucleosides and their analogs. The enzyme has been shown to have broad substrate specificity and plays a physiological role in the maintenance of the normal deoxyribonucleotide pools. Deoxycytidine kinase is also a key enzyme in the phosphorylation of a variety of antineoplastic and antiviral nucleoside analogs including 1-.beta.-D-arabinofuranosylcytosine and dideoxycytidine (Ullman, B. et al, J.Biol.Chem., 263: 12391-12396 (1988)), and deficiency of deoxycytidine kinase actively mediates resistance to these drugs. The enzyme is allosterically regulated by several deoxyribonucleotides and preferentially uses ATP as a phosphate donor for the phosphorylization of deoxycytidine (Ikeda, S. et al., Bio.Chem., 27: 8648-8652 (1988)).
Deoxycytidine kinase isolated from a human lymphoblastic cultured T-cell line had an apparent molecular weight of 60,000 and a Stokes radius of 32 .ANG.. It is thought that the deoxycytidine kinase enzyme is composed of two identical subunits. The rate of deoxycytidine phosphorylation is essentially greatest at pH 7.0 but there is a broad pH optimum ranging from pH 6.5 to pH 9.0. The enzyme also has an absolute requirement for magnesium. In the absence of added divalent cation, deoxycytidine is phosphorylated at about 10% of the rate seen with 2.4 mM magnesium.
An important structural function of the protein is the presence of a probable nucleotide binding domain at amino acids 28-34. The consensus sequence Gly-Xaa-Xaa-Gly-Xaa-Gly-Lys has been reported in several ATP-binding enzymes including a variety of thymidine kinases, the lactobacillus and AMP deaminase and deoxycytidine/deoxyadenosine kinase (Kim, M. et al., Bio.Chem.Biophys.Res.Commun., 156: 92-98 (1988)). The substitution of an alanine residue at position 31, does not prevent effective nucleotide binding. The deoxycytidine kinase protein has a number of substrates including cytosine arabinoside, deoxyguanosine, deoxyadenosine, cytidine, 2-chloro-adenosine and dideoxycytidine. If deoxycytidine kinase is contaminated with uridine-cytidine kinase, cytidine phosphorylation should be inhibited by uridine. While ATP is the preferred phosphate donor, GTP, ITP and XTP had more than 80% of activity as compared to ATP.
Deoxycytidine kinase has been partially purified from many sources (Baxter, A. et al., Bio.Chem.J., 173: 1005-1008 (1978)) including the following human tissues: tonsil lymphocytes, leukemia spleen, T-lymphoblasts, and myeloblasts.
Deoxycytidine kinase is the rate limiting step in the activation of the chemotherapeutic agent cytosine arabinoside to its 5' triphosphate (Mejer, J., Scand.J.Clin.Lab.Invest., 42: 401-406 (1982)). Other clinically important chemotherapeutic agents for which deoxycytidine kinase catalyzes the initial activation of is ara-C, 2-fluoro-9-.beta.-D-arabinofuranosyladenine, and dideoxycytidine (Kufe, D. W. and Spriggs, D. R., Semin.Oncol., 12: 34-48 (1985)).
The polypeptide of the present invention has been putatively identified as human deoxycytidine kinase 2. This identification has been made as a result of amino acid sequence homology to mouse deoxycytidine kinase 1.
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is hdCK2, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding hdCK2, including mRNAs, DNAs, cDNAs, genomic DNAs as well as an

REFERENCES:
Bohman et al., Deoxycytidine Kinase from Human Leukemic Spleen: Preparation and Characterization of the Homogeneous enzyme:, Biochemistry, vol. 27, issue 1988, pp. 4258-4265.
Chottiner et al, Cloning and Expression of Human Deoxycytidine Kinase cDNA: Proc. Natl. Acad. Sci. USA, vol. 88, Feb. 1991, pp. 1531-1535.
Song et al, "Genomic Structure and Chromosomal Localization of the Human Deoxycytidine Kinase Gene". Proc. Natl. Acad. Sci. USA, vol. 90, Jan. 1993, pp. 431-434.
Wang et al, "Purification and Characterization of Deoxycytidine Kinase from Acute Myeloid Leukemia Cell Mitochondria", Biochimica et Biophysica Acta, vol. 1202, Feb. 1993, pp. 309-316.
Eriksson et al., "Characterization of Human Deoxycytidine Kinase Correlation with cDNA Sequences", Federation of European Biochemical Societies, vol, 280, No. 2, Mar. 1991, pp. 363-366.
Cheng et al., "Human Deoxycytidine Kinase: Purification and Characterization of the Cytoplasmic and Mitochondrial Isozymes derived from Blast Cells of Acute Myelocytic Leukemia Patients" Biochimica et Biophysica Acta, vol, 481, 1977, pp. 481-492.
Huang et al., "Human Deoxycytidine Kinase Sequence of cDNA Clones and Analysis of Expression in Cell Lines with and without Enzyme Activity", Journal of Biological Chemistry, vol, 264, No. 25, Sep. 1989, pp. 14762-14768.
Johansson et al. Cloning and expression of human deoxyguanosine kinase cDNA. Proc. Nat. Acad. Sci., USA 93: 7258-7262, Jul. 1996.
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