Phosphorylation of deoxycytidine analog monophosphates by UMP-CMP kinase: molecular characterization of the human enzyme.
Phosphorylation of deoxycytidine analogs by cellular enzymes is a prerequisite for the activity of these compounds. We have investigated the kinetic parameters for the phosphorylation of 1-beta-D-arabinofuranosylcytosine (araC) and 2', 2'-difluorodeoxycytidine (dFdC) to their diphosphate forms catalyzed by human UMP-CMP kinase. We cloned the cDNA of this enzyme to enable characterization of the recombinant protein, determine its expression in different tissues, and determine the chromosome location of the gene. We showed that the recombinant UMP-CMP kinase phosphorylated CMP, dCMP, and UMP with highest efficiency and dUMP, AMP, and dAMP with lower efficiency. The monophosphates of araC and dFdC were shown to be phosphorylated with similar efficiency as dCMP and CMP. We further showed, in a combined enzymatic assay, that human deoxycytidine kinase and UMP-CMP kinase together phosphorylated araC, dFdC, and 2',3'-dideoxycytidine to their diphosphate forms. Northern blot analysis showed that the UMP-CMP kinase mRNA was ubiquitously present in human tissues as a 3.9-kb transcript with highest levels in pancreas, skeletal muscle, and liver. The human UMP-CMP kinase gene was localized to chromosome 1p34.1-1p33 by radiation hybrid analysis. We further expressed the UMP-CMP kinase as a fusion protein to the green fluorescent protein in Chinese hamster ovary cells, and showed that the fusion protein was located in the cytosol and nucleus.
Van Rompay
A R
AR
Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden.
Johansson
M
M
Karlsson
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A
D000595:Amino Acid Sequence; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D002874:Chromosome Mapping; D002878:Chromosomes, Human, Pair 1; D003001:Cloning, Molecular; D003561:Cytarabine / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D018076:DNA, Complementary; D011994:Recombinant Proteins; D003561:Cytarabine; D003841:Deoxycytidine; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D000093542:Gemcitabine