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 (kobs) 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, kobs 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 (KD) and maximum incorporation rate (kpol) determinations for dTTP and analogues lacking a 3′-OH indicated that low Mg2+ caused a several-fold greater reduction in kpol with the analogues but did not significantly affect KD, 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.