Synthese of Biotin Derivatives for Characterizing the Catalytic Mechanism of a Zn2+-Dependent Phosphorothiolate thiolesterase RNA

• Main Authors: Yi Chen, 陳毅
• Other Authors: Tzu-Pin Wang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/45655153182662478119

碩士 === 高雄醫學大學 === 醫藥暨應用化學研究所 === 97 === The purpose of this research is to decipher mechanistic properties essential to the catalysis of a phosphorothioate thiolesterase RNA that we previously in vitro isolated from a RNA pool. This phosphorothioate thiolesterase RNA is called the TW17 ribozyme. One of the exquisite properties of the TW17 ribozyme is the overwhelming requirement of zinc ions for its catalysis on phosphorothioate thiolester hydrolysis. The reaction is more efficient if Mg2+ ion participates in. We thus studied the kinetic behaviors of the TW17 ribozyme under the influence of pH and metal ion variations to fully perceive the catalytic mechanism of the TW17 ribozyme and its relevance to Zn2+-dependent protein hydrolases. Firstly, we measured the TW17 ribozyme-catalyzed pseudo-first-order rate constants of phosphorothioate thiolester hydrolysis under different pH conditions. The results indicated that the TW17 ribozyme activity followed a general base catalysis mechanism, and pKa = 7.0 ± 0.8. We also determined activities of the TW17 ribozyme in the presence of several different combinations of metal ions to demonstrate the dominant and positive effect of Zn2+ ion on catalysis. Finally, our acquired Zn2+ ion dissociation constant (Kd = 0.52 ± 0.03 mM at 0.1 M MgCl2; Kd = 0.58 ± 0.02 mM at no MgCl2) and the corresponding Hill coefficient (n = 3.88 at 0.1 M MgCl2; n = 4.36 at no MgCl2) for the TW17 ribozyme to unveil the dedicate interaction between the TW17 ribozyme and Zn2+ ion to confer this unique catalysis event. A similar study for the TW17 ribozyme and Mg2+ also revealed the vital role of Mg2+ on the TW17 ribozyme folding toward its maximal catalysis (Kd = 9.61 ± 1.59 mM; n = 2.75). These results shed light on the catalytic mechanism of TW17 ribozyme and may have significant implications for understanding the convergence of Zn2+-dependent hydrolysis catalyzed by ribozymes and protein enzymes.