NUCLEOSIDE TRIPHOSPHATE DONORS FOR NUCLEOSIDE KINASES - DONOR PROPERTIES OF UTP WITH HUMAN DEOXYCYTIDINE KINASE

The reported higher efficiency of UTP, relative to ATP, as phosphate d onor for deoxycytidine kinase (dCK), has been extended and found to ap ply to both dCyd and dAdo as accepters. UTP as phosphate donor was sho wn to follow strictly Michaelis kinetics, with K-m = 1 mu M, in striki ng contrast tb ATP, which exhibits marked negative cooperativity (Hill coef. = 0.7) with a several-fold higher K-m(app) = 15 mu M. Phosphate transfer was followed directly with use of mixtures of [gamma-P-32]AT P and cold UTP as donors, or with H-3-labeled accepters and cold donor s. With equimolar concentrations of ATP and UTP (50 mu M or 1 mM each) , and dCyd or dAdo as acceptor, only minimal phosphate transfer occurr ed from ATP (3-10%). With a 6:1 ratio of ATP:UTP, hence exceeding the intracellular ratio, phosphate transfer from ATP increased, but still did not exceed 25-40% with either dCyd or dAdo as acceptor. Moreover, relative ATP transfer is dependent on the dCyd concentration. We concl ude that the major intracellular phosphate donor for dCK is not ATP, b ut UTP. Preliminary data for human thymidine kinases (TK1 and TK2) exh ibit quite different behaviour. The foregoing, together with literatur e data, are highly relevant to in vitro studies on the properties of t he nucleoside kinases, and to the design of chemotherapeutically activ e nucleoside analogues. (C) 1995 Academic Press,Inc.