The mechanism of phosphorylation of anti-HIV D4T by nucleoside diphosphatekinase

The last step in the intracellular activation of antiviral nucleoside analo gs is the addition of the third phosphate by nucleoside diphosphate (NDP) k inase resulting in the synthesis of the viral reverse transcriptase substra tes. We have previously shown that dideoxynucleotide analogs and 3'-deoxy-3 '-azidothymidine (AZT) as di- or triphosphate are poor substrates for NDP k inase. By use of protein fluorescence, we monitor the phosphotransfer betwe en the enzyme and the nucleotide analog. Here, we have studied the reactivi ty of D4T (2',3'-dideoxy-2',3'-didehydrothymidine; stavudine) as di- (DP) o r triphosphate (TP) at the pre-steady state. The catalytic efficiency of D4 T-DP or -TP is increased by a factor of 10 compared with AZT-DP or -TP, res pectively. We use an inactive mutant of NDP kinase to monitor the binding o f a TP derivative, and show that the affinity for D4T-TP is in the same ran ge as for the natural substrate deoxythymidine triphosphate, but is 30 time s higher than for AZT-TP. Our results indicate that D4T should be efficient ly phosphorylated after intracellular maturation of a prodrug into D4T-mono phosphate.