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 analogs with various 3' sugar groups (-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). Analogs showed diminished incorporation rates (k) 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 analogs. Illustrating the importance of interactions between Mg2+ and the 3'-OH, k using 5 M dTTP and 0.5 mM Mg2+ was only modestly slower (1.6-fold) than with 6 mM Mg2+, while analogs with 3' alterations incorporated 2.8-5.1-fold slower in 0.5 mM Mg2+. In contrast, 4'-C-methyl and D-carb dTTP, which retain the 3'-OH, were not significantly affected by Mg2+. Consistent with these results, analogs with 3' modifications were better inhibitors in 6 mM vs. 0.5 mM Mg2+. Equilibrium dissociation constant (Kd) and kpol determinations for dTTP and analogs lacking a 3'-OH indicated that low Mg2+ caused a several-fold greater reduction in kpol with the analogs but had little effect on Kd, results consistent with a role for 3'-OH/Mg2+ interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance previously unreported interactions between Mg2+ and the 3'-OH of the incoming nucleotide and suggest inhibitors with 3'-OH groups may have advantages in the low free Mg2+ in physiological settings.