It has been generally accepted, on the basis of kinetic studies with phosphorothioate-containing substrates and analyses by NMR spectroscopy, that a divalent metal ion interacts directly with the pro-Rp oxygen at the cleavage site in reactions catalyzed by hammerhead ribozymes. However, results of our recent kinetic studies (Zhou, D.-M.; Kumar, P. K. R.; Zhang. L. H.; Taira, K. J. Am. Chem. Soc. 1996, 118, 8969-8970. Yoshinari, K.; Taira, K. Nucleic Acids Res. 2000, 28, 1730-1742) demonstrated that a Cd2+ ion does not interact with the sulfur atom at the Rp position of the scissile phosphate (P1.1) in the ground state or in the transition state. Therefore, in the present study, we attempted to determine by 31P NMR spectroscopy whether a Cd2+ ion binds to the P1.1 phosphorothioate at the cleavage site in solution. In the case of the R32-S11S (ribozyme-substrate) complex, neither the Rp- nor the Sp-phosphorothioate signal from the S11S substrate at the cleavage site was perturbed (the change was less than 0.1 ppm) upon the addition of Cd2+ ions (19 equiv) at pH 5.9 and 8.5. By contrast, we detected the significant perturbation of the P9 phosphorothioate signal from another known metal-binding site (the A9/G10.1 metal-binding motif). The Rp-phosphorothioate signal from A9/G10.1 was shifted by about 10 ppm in the higher field direction upon the addition of Cd2+ ions. These observations support the results of our kinetic analysis and indicate that a Cd2+ ion interacts with the sulfur atom of the phosphorothioate at the A9/G10.1 site (P9) but that a Cd2+ ion does not interact with the sulfur atom at the Rp- or at the Sp-position of the scissile phosphate (P1.1) in the ground state.
ASJC Scopus subject areas
- Colloid and Surface Chemistry