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.
Deoxythymidine triphosphate analogs with various 3’ groups (-OH (dTTP), -H, -N3, -NH2, -F, -O- CH3, and 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). All analogs showed diminished incorporation compared to dTTP ranging from about 2-fold (3’-H, -N3, and d4TTP with 6 mM Mg2+) to >10-fold (3’-NH2 and 3’-F with 0.5 mM Mg2+). The exception was 3’-O-CH3 dTTP which was incorporate profoundly more slowly than other analogs. The incorporation rate (k) using 5 µM dTTP and 0.5 mM (free) Mg2+ was modestly slower (1.6-fold) than with 6 mM Mg2+, while analogs with 3’ modifications were incorporated more slowly (2.8-5.1-fold) in 0.5 mM Mg2+. In contrast, 4’-C-methyl and D-carb, which retain the 3’-OH, were not significantly affected by Mg2+. Consistent with the above results, analogs with 3’ modifications were better inhibitors with 6 mM vs. 0.5 mM Mg2+, in primer extension reactions on a long template. 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. Overall, results emphasize the importance of as yet undefined interactions between Mg2+ and the 3’-OH and indicate that inhibitors with 3’-OH groups may have an advantage in a physiological setting where the concentration of free Mg2+ is low.
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