A reappraisal, based on (31)P NMR, of the direct coordination of a metal ion with the phosphoryl oxygen at the cleavage site of a hammerhead ribozyme.
A reappraisal, based on (31)P NMR, of the direct coordination of a metal ion with the phosphoryl oxygen at the cleavage site of a hammerhead ribozyme.
复制标题
基于 (31)P NMR 重新评估金属离子与锤头核酶裂解位点的磷酰氧的直接配位。
DOI：
10.1021/ja0202098
复制
发表时间：
2002
影响因子：
15
通讯作者：
K. Taira
中科院分区：
文献类型：
--
作者：
K. Suzumura;K. Yoshinari;Yoshiyuki Tanaka;Y. Takagi;Y. Kasai;M. Warashina;T. Kuwabara;M. Orita;K. Taira
基于含硫代磷酸酯底物的动力学研究和核磁共振波谱分析，人们普遍认为，在锤头核酶催化的反应中，二价金属离子直接与裂解位点的 pro-Rp 氧相互作用。然而，我们最近的动力学研究结果（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）表明Cd(2+) 离子不与基态或过渡态易裂磷酸盐 (P1.1) Rp 位置上的硫原子相互作用。因此，在本研究中，我们尝试通过(31)P NMR光谱确定Cd(2+)离子是否在溶液中的裂解位点与P1.1硫代磷酸酯结合。对于 R32-S11S（核酶-底物）复合物，在 pH 5.9 和 8.5 下添加 Cd(2+) 离子（19 当量）后，裂解位点处 S11S 底物的 Rp- 和 Sp-硫代磷酸酯信号均未受到干扰（变化小于 0.1 ppm）。相比之下，我们检测到来自另一个已知金属结合位点（A9/G10.1 金属结合基序）的 P9 硫代磷酸酯信号的显着扰动。添加 Cd(2+) 离子后，A9/G10.1 的 Rp-硫代磷酸酯信号在较高场方向上移动了约 10 ppm。这些观察结果支持我们的动力学分析结果，并表明 Cd(2+) 离子与 A9/G10.1 位点 (P9) 处的硫代磷酸酯的硫原子相互作用，但 Cd(2+) 离子不与基态下易裂磷酸盐 (P1.1) 的 Rp- 或 Sp-位置处的硫原子相互作用。
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 Cd(2+) 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 (31)P NMR spectroscopy whether a Cd(2+) 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 Cd(2+) 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 Cd(2+) ions. These observations support the results of our kinetic analysis and indicate that a Cd(2+) ion interacts with the sulfur atom of the phosphorothioate at the A9/G10.1 site (P9) but that a Cd(2+) 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.