Ohmichi T, Sugimoto N
Department of Chemistry, Faculty of Science, Konan University, Higashinada-ku, Kobe, Japan.
Biochemistry. 1997 Mar 25;36(12):3514-21. doi: 10.1021/bi962030d.
Leadzyme is a ribozyme that requires Pb2+. We have previously shown that the addition of Nd3+ in the presence of Pb2+ increased significantly the yield of the RNA cleavage reaction by a leadzyme, although other rare earth ions or divalent ions except Pb2+ did not promote the reaction [Sugimoto, N., & Ohmichi, T. (1996) FEBS Lett. 393, 97-100]. To investigate the combined effects of Nd3+ and Pb2+ on the binding and cleavage steps of a leadzyme, CUGGGAGUCC, with a substrate, GGACCGAGCCAG, kinetics for the leadzyme reaction have been measured at various concentration ratios of Nd3+ and Pb2+. At low concentration ratios of Nd3+ under a constant total concentration of metal ions, Nd3+ increased the stability of the complex between the leadzyme and the substrate. In contrast, at high concentration ratios of Nd3+, the addition of Nd3+ decreased the stability of the complex. The rate constant of the cleavage step was maximized when the ratio of Nd3+ to Pb2+ was 1:1. These results suggest that the complex between the leadzyme and the substrate has binding sites for Nd3+ ion that influence complex stability and catalyze directly the cleavage reaction. On the basis of the results, we propose a two-metal-ion mechanism in which Pb2+ and Nd3+ play the roles of base and acid catalyst, respectively.
铅酶是一种需要Pb2+的核酶。我们之前已经表明，在Pb2+存在的情况下添加Nd3+会显著提高铅酶催化的RNA切割反应的产率，尽管除Pb2+之外的其他稀土离子或二价离子并不能促进该反应[Sugimoto, N., & Ohmichi, T. (1996) FEBS Lett. 393, 97 - 100]。为了研究Nd3+和Pb2+对铅酶CUGGGAGUCC与底物GGACCGAGCCAG结合和切割步骤的联合作用，我们在不同的Nd3+和Pb2+浓度比下测量了铅酶反应的动力学。在金属离子总浓度恒定的情况下，当Nd3+浓度比很低时，Nd3+会增加铅酶与底物之间复合物的稳定性。相反，当Nd3+浓度比很高时，添加Nd3+会降低复合物的稳定性。当Nd3+与Pb2+的比例为1:1时，切割步骤的速率常数最大。这些结果表明，铅酶与底物之间的复合物具有Nd3+离子的结合位点，这些位点会影响复合物的稳定性并直接催化切割反应。基于这些结果，我们提出了一种双金属离子机制，其中Pb2+和Nd3+分别扮演碱催化剂和酸催化剂的角色。
