Lott W B, Pontius B W, von Hippel P H
Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.
Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):542-7. doi: 10.1073/pnas.95.2.542.
Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.
通过在恒定浓度的Mg2+存在下监测RNA底物的切割速率随La3+浓度的变化，为HH16锤头状核酶的双金属离子切割机制提供了证据。我们发现，在8 mM Mg2+存在下，以微摩尔浓度添加La3+时，会得到一条切割活化的钟形曲线，在3 μM La3+存在下达到最大切割速率。这些结果表明，核酶上的双金属离子结合位点调节切割反应的速率，并且根据先前对Mg2+的Kd值估计为3.5 mM和> 50 mM，这些位点结合La3+的估计Kd值分别为0.9和> 37.5 μM。此外，鉴于这些金属离子在两个结合位点的作用非常不同，在较强（相对于Mg2+）结合位点上La3+取代Mg2+会激活催化作用，而在较弱（相对于Mg2+）（相对于Mg2+）结合位点上La3+取代Mg2+会抑制催化作用，我们表明这两个位点的金属离子起着非常不同的作用。我们认为，结合位点1的金属离子在反应中协调进攻的2'-氧物种，并降低所连接质子的pKa，从而在平衡过程中增加进攻醇盐亲核试剂的浓度。相比之下，结合位点2的金属离子的作用是通过吸收在过渡态离开的5'-氧上积累的负电荷来催化反应。我们提出了结构上的原因，解释了为什么Mg(2+)-La3+离子组合特别适合证明双金属离子在核酶切割反应中的这些不同作用。
