Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme

The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn(2+) ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na(+) ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses.

锤头核酶（hammerhead ribozyme）已被普遍认定为一种特征明晰的金属酶。然而，RNA与金属离子相互作用的精确本质仍有待全面阐明。在有限金属离子浓度下考察金属离子催化的锤头核酶反应，有助于评估金属离子的作用，本研究即证实了这一结论。当Mn²⁺离子浓度处于0.3至3 mM范围内时，在单周转（single-turnover）条件下向反应体系中加入核酶可促进反应，产物最终达到固定的最大水平。若继续追加核酶，则会抑制反应，这表明核酶的正确折叠与催化过程均需要一定量的二价金属离子。在极高浓度下，一价金属离子（如Na⁺）也可作为锤头核酶催化反应的辅助因子，但其催化效率极低，因此需要极高浓度才能发挥作用。此外，向二价金属离子催化的锤头核酶反应体系中加入一价离子，会抑制二价金属离子介导的催化过程，这提示更具活性的二价金属离子-核酶复合物，会通过移除作为核酶复合物结构支撑的二价金属离子，转化为活性较低的一价金属离子-核酶复合物。尽管存在两条催化途径：即高效的二价金属离子依赖型途径与低效的一价金属离子依赖型途径，但显然在生理条件下，锤头核酶作为金属酶，主要通过催化效率显著更高的二价金属离子依赖途径发挥功能。此外，本研究观测到的动力学数据，与Lilley及DeRose基于基态结构分析提出的两相折叠模型相一致。