Phosphorylation of deoxycytidine analog monophosphates by UMP-CMP kinase: Molecular characterization of the human enzyme

Phosphorylation of deoxycytidine analogs by cellular enzymes is a prerequis ite for the activity of these compounds. We have investigated the kinetic p arameters for the phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara C) and 2',2'-difluorodeoxycytidine (dFdC) to their diphosphate forms cataly zed by human UMP-CMP kinase. We cloned the cDNA of this enzyme to enable ch aracterization of the recombinant protein, determine its expression in diff erent 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 mono phosphates of araC and dFdC were shown to be phosphorylated with similar ef ficiency 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, s keletal muscle, and liver. The human UMP-CMP kinase gene was localized to c hromosome 1p34.1-1p33 by radiation hybrid analysis. We further expressed th e UMP-CMP kinase as a fusion protein to the green fluorescent protein in Ch inese hamster ovary cells, and showed that the fusion protein was located i n the cytosol and nucleus.