Fig. 3. Enhancement of AZT and d4T activation by NDPK in the presence of the R_p α-borano group. (A) Kinetics of phosphorylation of AZT diphosphate and α-borano-AZT diphosphate by phosphorylated human NDPK A. The phosphorylated enzyme (1 µM), prepared as described (Deville-Bonne et al., 1996), is mixed with 400 µM AZT diphosphate or α-borano-AZT diphosphate in 50 mM Tris–HCl pH 7.5, 5 mM MgCl₂, 75 mM KCl, 1 mM DTT and 5% glycerol (final concentrations) at 20°C. The decrease in fluorescence is monitored with a Hi-Tech SF-61DX2 stopped flow device (λ_exc = 304 nm, excitation slit = 2 nm, emission filter <320 nm). The solid lines represent the best fit of each curve to a monoexponential. (B) Concentration dependence of the pseudo-first order phosphorylation rate constant k_obs: α-borano-AZT diphosphate (circles) and AZT diphosphate (squares). Data were analysed according to the classical model of a fast binding reaction followed by a slow, rate-limiting phosphotransfer (Schneider et al., 1998). The catalytic efficiency is k_max/K_D = 2000 M^–1s^–1 for α-borano-AZT diphosphate and 200 M^–1s^–1 for AZT diphosphate. As no saturation is reached, the maximum rate of phosphotransfer, k_max, is not measurable in this experiment. (C) Concentration dependence of the pseudo-first order rate constant k_obs for NDPK A phosphorylation by α-borano-d4T triphosphate (diamonds) and d4T triphosphate (triangles). The catalytic efficiency is k_max/K_D = 6000 M^–1s^–1 for α-borano-d4T triphosphate and 800 M^–1s^–1 for d4T triphosphate.