A KINETIC MECHANISM FOR CLEAVAGE OF PRECURSOR TRNA(ASP) CATALYZED BY THE RNA COMPONENT OF BACILLUS-SUBTILIS RIBONUCLEASE-P
A KINETIC MECHANISM FOR CLEAVAGE OF PRECURSOR TRNA(ASP) CATALYZED BY THE RNA COMPONENT OF BACILLUS-SUBTILIS RIBONUCLEASE-P
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DOI：
10.1021/bi00200a009
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发表时间：
1994-08-30
期刊：
影响因子：
2.9
通讯作者：
FIERKE, CA
中科院分区：
文献类型：
--
作者：
BEEBE, JA;FIERKE, CA
提出了B的RNA组分催化枯草芽孢杆菌前体tRNA（Asp）裂解的动力学机制。枯草杆菌核糖核酸酶P（RNase P）在最佳条件（50 mM Tris Cl（pH 8.0）、100 mM MgCl 2和800 mM NH 4Cl，37 ℃）下进行。该动力学机制来自于使用猝灭流、凝胶过滤和凝胶移位技术的组合测量预稳态、稳态、单周转和结合动力学。一个最小的动力学描述涉及以下内容：（1）前tRNA（Asp）与RNase P RNA的快速结合（6.3 × 10（6）M（-1）s（-1）），但低于扩散控制极限;（2）磷酸二酯键断裂，速率常数为6 s（-1）;（3）产物在动力学优选途径中的解离，5 ′ RNA片段首先解离（大于或等于0.2 s（-1）），随后是限速tRNA解离（0.02 s（-1））;和（4）在催化循环过程中形成RNase P RNA的第二构象异构体，其不太稳定并且显著更慢地结合前-tRNA（Asp（7 × 10（4）M（-1）s（-1））。该方案涉及反应序列中各个步骤的分离，与稳态数据一致，并在各种条件下精确定位速率决定步骤。这种动力学机制将有助于更准确地定义金属，pH值和蛋白质组分在反应的每一步中的作用，并为理解结构变化对催化活性的影响提供必要的背景。
A kinetic mechanism is presented for the cleavage of Bacillus subtilis precursor tRNA(Asp) catalyzed by the RNA component of B. subtilis ribonuclease P (RNase P) under optimal conditions (50 mM Tris Cl (pH 8.0), 100 mM MgCl2, and 800 mM NH4Cl, 37 degrees C). This kinetic mechanism was derived from measuring pre-steady-state, steady-state, single-turnover, and binding kinetics using a combination of quench-flow, gel filtration, and gel shift techniques. A minimal kinetic description involves the following: (1) binding of pre-tRNA(Asp) to RNase P RNA rapidly (6.3 x 10(6) M(-1) s(-1)), but slower than the diffusion-controlled limit; (2) cleavage of the phosphodiester bond with a rate constant of 6 s(-1); (3) dissociation of products in a kinetically preferred pathway, with the 5' RNA fragment dissociating first (greater than or equal to 0.2 s(-1)) followed by rate-limiting tRNA dissociation (0.02 s(-1)); and (4) formation of a second conformer of RNase P RNA during the catalytic cycle that is less stable and binds pre-tRNA(Asp) significantly more slowly (7 X 10(4) M(-1) s(-1)). This scheme involves the isolation of individual steps in the reaction sequence, is consistent with steady-state data, and pinpoints the rate-determining step under a variety of conditions. This kinetic mechanism will facilitate a more accurate definition of the role of metals, pH, and the protein component in each step of the reaction and provide an essential background for understanding the influence of structural changes on the catalytic activity.