SUBSTRATE-SPECIFICITY OF MITOCHONDRIAL 2'-DEOXYGUANOSINE KINASE - EFFICIENT PHOSPHORYLATION OF 2-CHLORODEOXYADENOSINE

Mitochondrial deoxyguanosine kinase (dGK) (EC 2.7.1.113) was purified to apparent homogeneity from bovine brain. The molecular mass of the n ative protein was 56 kDa, as judged by gel filtration, and one single band of 28 kDa was seen in sodium dodecyl sulfate-gel electrophoresis. 2'-Deoxyguanosine (dGuo) (K(m), 7.6 muM), 2'-deoxyinosine, and 2'-deo xyadenosine (K(m), 60 muM) were substrates for the enzyme as well as s everal dGuo analogs containing a lipophilic substituent at C-2'. Carbo cyclic dGuo, 9-beta-D-arabinofuranosylguanine, 9-beta-D-arabinofuranos ylhypoxanthine, and 9-beta-D-arabinofuranosyladenine were substrates f or the enzyme, whereas no 3'-modified dGuo analogs were effective. Int erestingly, 2-chloro-2'-deoxyadenosine (CdA) was found to be an effici ent substrate for dGK (K(m), 85 muM). Subcellular fractionation of hum an CEM lymphoblasts showed that extracts of mitochondria contain signi ficant CdA phosphorylating activity (71.5 pmol/mg/min) that is not inh ibited by excess of 2'-deoxycytidine (dCyd). This contrasts with the C dA phosphorylating activity found in cytosolic extracts, which is carr ied out by dCyd kinase and strongly inhibited by excess of dCyd. The e fficient CdA phosphorylation by mitochondrial dGK is a novel finding t hat may have far reaching implications for the clinical use of this po tent cytostatic drug.