Inorganic tripolyphosphate (PPPi) and pyrophosphate (PPi) were examined as potential phosphate donors for human deoxynucleoside kinase (dCK), deoxyguanosine kinase (dGK), cytosolic thymidine kinase (TK1), mitochondrial TK2, and the deoxynucleoside kinase (dNK) from Drosophila melanogaster. PPPi proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not dAdo, as acceptor substrate, illustrating also the dependence of donor properties on acceptor. Products of phosphorylation were shown to be 5'-phosphates. In striking contrast to ATP, the phosphorylation reaction follows strict Michaelis-Menten kinetics, with K-m values of 74 and 92 muM for dCK and dGK, respectively, and V-max values 40-50% that for ATP. With the other three enzymes, as well as for dCK with dAdo as acceptor, no, or only low levels (less than or equal to1% of that for ATP) of activity were observed. PPi was inactive (<0.1%) as a phosphate donor with all enzymes, but was a competitive inhibitor vs ATP, as was PPPi in systems with no or low donor activity. This is the first report on inorganic tripolyphosphate as a phosphate donor for nucleoside kinases, in particular human deoxyribonucleoside kinases. (C) 2003 Elsevier Science (USA). All rights reserved.