McConnell T S, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1995 Mar 28;34(12):4056-67. doi: 10.1021/bi00012a024.
The ribozyme derived from the group I intron of Tetrahymena thermophila binds an exogenous guanosine nucleotide, which acts as the nucleophile in the sequence-specific cleavage of oligonucleotides. By examining the temperature dependence of the reaction under conditions where Km = Kd, we conclude the following: (1) Guanosine 5'-monophosphate (pG) binds to the closed ribozyme-oligonucleotide substrate complex with a positive entropy change (delta S degree' = +23 eu) and an enthalpy change (delta H degree') close to zero. This is contrary to the expectation that binding would cause increased order (negative delta S degree) and be driven by a negative delta H degree. (2) Inosine and 2-aminopurine riboside, each lacking two hydrogen-bonding moieties relative to guanosine, also bind with a positive entropy value and an unfavorable (positive) delta H degree'. From this result, we suggest that the hydrogen-bonding moieties make an enthalpic contribution to guanosine binding overcoming an intrinsic unfavorable delta H. (3) At 0 degree C, there is equally tight binding of pG in the presence and absence of oligonucleotide substrate bound to the ribozyme. Thus, energetic interactions responsible for the thermodynamic coupling between pG and oligonucleotide substrate binding seen at higher temperatures are indirect. (4) The activation barrier of the chemical step is stabilized by a positive delta S++ (+31 to 39 eu). This stabilization is seen in four reactions using substrates with two different leaving groups in the presence and absence of pG, suggesting that the entropic contribution is inherent to the active site. The positive delta S values for the chemical step and for the binding of pG can be explained by a conformational change or release of water. Thus, although hydrogen bonding contributes to binding of nucleotides to this RNA enzyme as previously thought, it is these other events which produce a positive delta S that provide the energetic driving force for binding.
源自嗜热四膜虫I组内含子的核酶结合一种外源性鸟苷酸，该鸟苷酸在寡核苷酸的序列特异性切割中作为亲核试剂。通过在Km = Kd的条件下研究反应的温度依赖性，我们得出以下结论：（1）5'-鸟苷单磷酸（pG）以正熵变（ΔS°' = +23 eu）和接近零的焓变（ΔH°'）结合到闭合的核酶 - 寡核苷酸底物复合物上。这与预期相反，即结合会导致有序性增加（负ΔS°）并由负ΔH°驱动。（2）相对于鸟苷各自缺少两个氢键部分的肌苷和2-氨基嘌呤核糖苷，也以正熵值和不利的（正的）ΔH°'结合。从这个结果，我们认为氢键部分对鸟苷结合有焓贡献，克服了内在的不利ΔH。（3）在0℃时，在存在和不存在与核酶结合的寡核苷酸底物的情况下，pG的结合同样紧密。因此，在较高温度下看到的pG与寡核苷酸底物结合之间的热力学偶联所涉及的能量相互作用是间接的。（4）化学步骤的活化能垒通过正ΔS++（+31至39 eu）而稳定。在使用具有两种不同离去基团的底物且存在和不存在pG的四个反应中都观察到这种稳定化，表明熵贡献是活性位点所固有的。化学步骤和pG结合的正ΔS值可以通过构象变化或水的释放来解释。因此，尽管如先前认为的那样氢键有助于核苷酸与这种RNA酶的结合，但正是这些产生正ΔS的其他事件为结合提供了能量驱动力。
