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

Abstract

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+ , Mn 2+ , and Co 2+ . HIV‐1 RT exhibits a strong preference to incorporate Sp‐TTPαS over Rp‐TTPαS 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‐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.