The first pre-steady-state kinetic analysis of the stereo-selective incorporation of Rp- and Sp-isomers of thymidine-5′-O-1-thiotriphosphate (TTPαS) by HIV-1 reverse transcriptase (RT) is reported. Rates of polymerization (kpol), apparent dissociation constants (K d), and substrate specificities (kpol/Kd) were measured for TTP, Rp-TTPαS, and Sp-TTPαS in the presence of Mg 2+, Mn2+, and Co2+. HIV-1 RT exhibits a strong preference to incorporate Sp-TTPαS over Rp-TTPαS in the presence of Mg2+; however, this stereo-selective preference was decreased when Mg2+ was replaced with Mn2+ and Co2+. Furthermore, HIV-1 RT exhibited no phosphorothioate elemental effects for the incorporation of Sp-TTPαS, but large elemental effects were calculated for Rp-TTPαS for each of the metals tested. These results are discussed in relation to our current understanding of the RT active-site structure and the mechanism of DNA synthesis.
All Science Journal Classification (ASJC) codes
- Molecular Biology