PHOSPHORYLATION OF ANTICANCER NUCLEOSIDE ANALOGS BY HUMAN MITOCHONDRIAL DEOXYGUANOSINE KINASE

The kinetic properties of recombinant human mitochondrial deoxyguanosi ne kinase (dGK, EC 2.7.1.113) for 2'-deoxyguanosine and the clinically important nucleoside analogs 2-chloro-2'-deoxyadenosine (CdA), 9-beta -D-arabinofuranosylguanine (araG) and 2',2',-difluorodeoxyguanosine ( dFdG) were determined. The Michaelis-Menten kinetic parameters, compar ing ATP and UTP as phosphate donors, demonstrated a marked increase in phosphorylation efficiency (VmaxKm) with UTP in comparison with ATP f or both CdA and araG. The difluoro analog dFdG was an efficient substr ate for recombinant dGK with an apparent K-m of 16 mu M with ATP as ph osphate donor. We compared the kinetic properties of dGK with those of the related enzyme deoxycytidine kinase (dCK, EC 2.7.1.74). Although the purines 2'-deoxyguanosine (dGuo) and 2'-deoxyadenosine are substra tes for both dGK and dCK, only CdA among the purine nucleoside analogs tested was an efficient substrate for both dCK and dGK. In competitio n with dGuo, the most efficient analog for phosphorylation by dGK was araG, as indicated by a lower K-i value than for CdA and dFdG. Of the purine analogs tested as substrates for dCK, only CdA. could compete w ith 2'-deoxycytidine (dCyd). No inhibition of dCK-mediated dCyd phosph orylation was found by either araG or dFdG. In crude cell extract of H eLa and Capan 2 cells, the major CdA phosphorylation was contributed b y dCK, while most araG phosphorylation was a result of dGK activity. O ur study with pure recombinant enzymes confirms that dGK is mainly res ponsible for araG and dFdG phosphorylation, whereas dCK is the most im portant enzyme for activation of CdA and 2',2'-difluorodeoxycytidine ( dFdC). (C) 1998 Elsevier Science Inc.