Substrate Specificity of Human Recombinant Mitochondrial Deoxyguanosine Kinase with Cytostatic and Antiviral Purine and Pyrimidine Analogs

Abstract

Deoxyguanosine kinase (dGK) is an enzyme responsible for the phosphorylation of purine deoxynucleosides in mitochondria of mammalian cells. Its role in activation of pharmacologically used nucleoside analogs is not well understood, because of the low levels of dGK found in tissue extracts and its inactivation during purification. The cDNA for dGK was recently cloned and expressed in Escherichia coli. Here we present an improved procedure for expression and purification of a highly active form of human recombinant dGK. The enzyme showed a broad substrate specificity toward natural purine and pyrimidine deoxynucleosides as well as toward important nucleoside analogs. The K m and V max values for deoxyguanosine, deoxyinosine, deoxyadenosine, and deoxycytidine were 4, 13, 460, 330 M and 43, 330, 430 and 60 nmol/min/mg of protein,

Mechanisms of anti-cancer action and pharmacology of clofarabine

“…Upon entry into cells, clofarabine is phosphorylated to its monophosphate derivatives, primarily by dCK, a constitutively expressed key cytosolic enzyme involved in the salvage pathway of DNA synthesis, but also by the mitochondrial enzyme deoxyguanosine kinase (dGK) [17]. Further intracellular phosphorylation results in the final metabolite, clofarabine triphosphate.…”

Nucleoside analogues: mechanisms of drug resistance and reversal strategies

“…This enzyme showed a broad substrate specificity towards natural purine deoxynucleosides, as well as towards nucleoside analogues such as ara-G and 2-CdA. 55,73,92 dGK is located predominantly in the mitochondria. 131 However, Petrakis et al 132 described a novel aminoterminally truncated isoform that corresponds to about 14% of the total dGK mRNA population in mouse spleen.…”

“…69 The different effects of F-ara-A and 2-CdA on mitochondria integrity may be related to the fact that 2-CdATP may accumulate more rapidly than F-ara-ATP in the mitochondria because it is converted with higher efficiency by mitochondrial dGK. 92 It has also been stated that 2-chloro derivatives are slightly more hydrophobic than the corresponding 2-fluoro derivatives and therefore may be able to insert more easily into the mitochondrial membrane, thereby affecting their integrity. 69 In summary inhibition of DNA repair, termination of mRNA transcription and the consequent depletion of proteins required for cell survival, as well as the capacity of F-ara-ATP to activate the apoptosome pathway appear to contribute to the cytotoxicity of fludarabine in non-dividing cells.…”

Real-time quantitative PCR assays for deoxycytidine kinase, deoxyguanosine kinase and 5’-nucleotidase mRNA measurement in cell lines and in patients with leukemia

“…The mitochondrial enzyme deoxyguanosine kinase (dGK) phosphorylates several important nucleoside analogues. 1 Both purine and pyrimidine analogues can be dephosphorylated by members of the 5′-nucleotidase (5′-NT) enzyme family and by non-specific phosphatases. The low-K m 5′-NT dephosphorylates pyrimidine monophosphates and the high-K m 5′-NT is specific for purine ribonucleotides.…”