McConnell T S, Herschlag D, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Biochemistry. 1997 Jul 8;36(27):8293-303. doi: 10.1021/bi9700678.
The Tetrahymena thermophila L-21 ScaI ribozyme utilizes Mg2+ to catalyze a site-specific endonuclease reaction analogous to the first step of self-splicing. To better understand the contribution of Mg2+ to ribozyme activity, the Mg2+ concentration dependence of individual rate constants was examined at concentrations greater than those required for ribozyme folding (>2 mM; at 50 degrees C and pH 6.7). Analysis of metal ion inhibition of the chemical step of the reaction indicated that two Ca2+ ions compete with two Mg2+ ions involved in active site chemistry. These Mg2+ ions are bound tightly to the E.S complex (Kd < 2 mM). The rate constant for association of the oligoribonucleotide substrate (S) increased 12-fold from 2 to 100 mM Mg2+ and exhibited saturation behavior, consistent with a single Mg2+ ion involved in S association that binds to the free ribozyme with a Kd for Mg2+ of 15 mM. The preference for the divalent metal ion (Mg2+ congruent with Ca2+ > Ba2+ >> Sr2+) suggested that enhancing the rate constant of S association is not simply a function of ionic strength, but is due to a distinct metal ion binding site. Even though Ca2+ does not support reaction, the RNA substrate S was able to bind in the presence of Ca2+. Upon addition of Mg2+, S was cleaved without first dissociating. A model is proposed in which the inactive Ca2+ form of E.S is structurally equivalent to the open complex along the reaction pathway, which has the RNA substrate bound but not docked into the active site. Weaker binding of S in Ca2+ was shown to result from an increase in the rate constant of S dissociation, leading to the proposal that a tight Mg2+ binding site or sites in the E.S complex contribute to the strong binding of S. In summary, the data provide evidence for four functions for bound Mg2+ ions in the catalytic cycle: one increases the rate of RNA substrate binding, one or more decrease the rate of dissociation of S, and two are involved in the chemical step.
嗜热四膜虫L-21 ScaI核酶利用Mg2+催化位点特异性内切核酸酶反应，该反应类似于自我剪接的第一步。为了更好地理解Mg2+对核酶活性的贡献，在高于核酶折叠所需浓度（>2 mM；50℃和pH 6.7）的浓度下，研究了各个速率常数对Mg2+浓度的依赖性。对反应化学步骤的金属离子抑制分析表明，两个Ca2+离子与参与活性位点化学作用的两个Mg2+离子竞争。这些Mg2+离子与E.S复合物紧密结合（Kd<2 mM）。寡核糖核苷酸底物（S）的缔合速率常数在Mg2+浓度从2 mM增加到100 mM时增加了12倍，并表现出饱和行为，这与参与S缔合的单个Mg2+离子一致，该离子以15 mM的Mg2+解离常数（Kd）与游离核酶结合。对二价金属离子的偏好（Mg2+≈Ca2+>Ba2+>>Sr2+）表明，提高S缔合的速率常数不仅仅是离子强度的函数，而是由于一个独特的金属离子结合位点。尽管Ca2+不支持反应，但RNA底物S在Ca2+存在下仍能结合。加入Mg2+后，S在未先解离的情况下被切割。提出了一个模型，其中E.S的无活性Ca2+形式在结构上等同于反应途径中的开放复合物，该复合物结合了RNA底物但未对接至活性位点。结果表明，S在Ca2+中的较弱结合是由于S解离速率常数增加所致，这表明E.S复合物中一个或多个紧密的Mg2+结合位点有助于S的强结合。总之，这些数据为催化循环中结合的Mg2+离子的四种功能提供了证据：一种功能增加RNA底物结合速率，一种或多种功能降低S的解离速率，还有两种功能参与化学步骤。
