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

Abstract

Deoxythymidine triphosphate analogues with various 3' substituents in the sugar ring (-OH (dTTP)), -H, -N₃, -NH₂, -F, -O-CH₃, no group (2',3'-didehydro-2',3'-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3'-OH but with 4' additions (4'-C-methyl, 4'-C-ethyl) or sugar ring modifications (d-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg²⁺ with HIV reverse transcriptase (RT). Analogues showed diminished observed incorporation rate constants (k _obs) compared to dTTP ranging from about 2-fold (3'-H, -N₃, and d4TTP with high Mg²⁺) to >10-fold (3'-NH₂ and 3'-F with low Mg²⁺), while 3'-O-CH₃ dTTP incorporated much slower than other analogues. Illustrating the importance of interactions between Mg²⁺ and the 3'-OH, k _obs using 5 μM dTTP and 0.5 mM Mg²⁺ was only modestly slower (1.6-fold) than with 6 mM Mg²⁺, while analogues with 3' alterations incorporated 2.8-5.1-fold slower in 0.5 mM Mg²⁺. In contrast, 4'-C-methyl and d-carba dTTP, which retain the 3'-OH, were not significantly affected by Mg²⁺. Consistent with these results, analogues with 3' modifications were better inhibitors in 6 versus 0.5 mM Mg²⁺. Equilibrium dissociation constant (K _D) and maximum incorporation rate (k _pol) determinations for dTTP and analogues lacking a 3'-OH indicated that low Mg²⁺ caused a several-fold greater reduction in k _pol with the analogues but did not significantly affect K _D, results consistent with a role for 3'-OH/Mg²⁺ interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance of previously unreported interactions between Mg²⁺ and the 3'-OH of the incoming nucleotide and suggest that inhibitors with 3'-OH groups may have advantages in low free Mg²⁺ in physiological settings.