HIV Reverse Transcriptase Pre-Steady-State Kinetic Analysis of Chain Terminators and Translocation Inhibitors Reveals Interactions between Magnesium and Nucleotide 3'-OH.

Deoxythymidine triphosphate analogues with various 3' substituents in the sugar ring (-OH (dTTP)), -H, -N, -NH, -F, -O-CH, no group (2',3'-didehydro-2',3'-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3'-OH but with 4' additions (4'--methyl, 4'--ethyl) or sugar ring modifications (d-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg with HIV reverse transcriptase (RT). Analogues showed diminished observed incorporation rate constants ( ) compared to dTTP ranging from about 2-fold (3'-H, -N, and d4TTP with high Mg) to >10-fold (3'-NH and 3'-F with low Mg), while 3'-O-CH dTTP incorporated much slower than other analogues. Illustrating the importance of interactions between Mg and the 3'-OH, using 5 μM dTTP and 0.5 mM Mg was only modestly slower (1.6-fold) than with 6 mM Mg, while analogues with 3' alterations incorporated 2.8-5.1-fold slower in 0.5 mM Mg. In contrast, 4'--methyl and d-carba dTTP, which retain the 3'-OH, were not significantly affected by Mg. Consistent with these results, analogues with 3' modifications were better inhibitors in 6 versus 0.5 mM Mg. Equilibrium dissociation constant ( ) and maximum incorporation rate ( ) determinations for dTTP and analogues lacking a 3'-OH indicated that low Mg caused a several-fold greater reduction in with the analogues but did not significantly affect , results consistent with a role for 3'-OH/Mg interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance of previously unreported interactions between Mg and the 3'-OH of the incoming nucleotide and suggest that inhibitors with 3'-OH groups may have advantages in low free Mg in physiological settings.