Stereo-selectivity of HIV-1 reverse transcriptase toward isomers of thymidine-5′-O-1-thiotriphosphate

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 (k_pol), apparent dissociation constants (K _d), and substrate specificities (k_pol/K_d) 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.