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

[Image: see text] Deoxythymidine triphosphate analogues with various 3′ substituents in the sugar ring (−OH (dTTP)), −H, −N(3), −NH(2), −F, −O–CH(3), 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) Mg(2+) with HIV reverse transcriptase (RT). Analogues showed diminished observed incorporation rate constants (k(obs)) compared to dTTP ranging from about 2-fold (3′-H, −N(3), and d4TTP with high Mg(2+)) to >10-fold (3′-NH(2) and 3′-F with low Mg(2+)), while 3′-O-CH(3) dTTP incorporated much slower than other analogues. Illustrating the importance of interactions between Mg(2+) and the 3′-OH, k(obs) using 5 μM dTTP and 0.5 mM Mg(2+) was only modestly slower (1.6-fold) than with 6 mM Mg(2+), while analogues with 3′ alterations incorporated 2.8–5.1-fold slower in 0.5 mM Mg(2+). In contrast, 4′-C-methyl and d-carba dTTP, which retain the 3′-OH, were not significantly affected by Mg(2+). Consistent with these results, analogues with 3′ modifications were better inhibitors in 6 versus 0.5 mM Mg(2+). Equilibrium dissociation constant (K(D)) and maximum incorporation rate (k(pol)) determinations for dTTP and analogues lacking a 3′-OH indicated that low Mg(2+) 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/Mg(2+) interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance of previously unreported interactions between Mg(2+) and the 3′-OH of the incoming nucleotide and suggest that inhibitors with 3′-OH groups may have advantages in low free Mg(2+) in physiological settings.