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 (TTPalphaS) 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-TTPalphaS, and Sp-TTPalphaS in the presence of Mg(2+), Mn(2+), and Co(2+). HIV-1 RT exhibits a strong preference to incorporate Sp-TTPalphaS over Rp-TTPalphaS in the presence of Mg(2+); however, this stereo-selective preference was decreased when Mg(2+) was replaced with Mn(2+) and Co(2+). Furthermore, HIV-1 RT exhibited no phosphorothioate elemental effects for the incorporation of Sp-TTPalphaS, but large elemental effects were calculated for Rp-TTPalphaS 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.