Three critical hydrogen bonds determine the catalytic activity of the Diels-Alderase ribozyme.
Three critical hydrogen bonds determine the catalytic activity of the Diels-Alderase ribozyme.
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DOI：
10.1093/nar/gkr812
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发表时间：
2012-02
影响因子：
14.9
通讯作者：
Jäschke A
中科院分区：
文献类型：
--
作者：
Kraut S;Bebenroth D;Nierth A;Kobitski AY;Nienhaus GU;Jäschke A
与蛋白质酶相比，我们对RNA如何加速化学反应的了解相当有限。催化Diels-Alder反应的核酶的晶体结构表明具有丰富的三级结构。在这项研究中，我们使用原子诱变与各种分析技术相结合，系统地探讨了晶体学观察到的基态相互作用与催化功能的相关性。最大的能量贡献显然来自于过渡态和催化口袋之间精确的形状互补：一个单点突变体正确折叠成三级结构，但缺乏一个通常稳定口袋的氢键，完全不活跃。在限速的化学步骤中，二亲试剂进一步被两个弱氢键激活，这两个弱氢键对过渡态稳定的贡献为~ 7-8 kJ/mol，正如缺失突变体的反应速度慢25倍所表明的那样。这些氢键也负责核酶与Diels-Alder产物的紧密结合，从而导致产物抑制。为了获得高的催化活性，核酶需要在刚性和柔韧性之间保持良好的平衡，这是由一个链间氢键和一个镁离子的联合作用决定的。在tRNA中观察到的与T-loop motif相似的360°急转弯被发现对催化功能很重要。
Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function.
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影响因子：
56.9
作者：
Chworos, A;Severcan, I;Jaeger, L
通讯作者：
Jaeger, L
影响因子：
16.6
作者：
Kobitski, Andrei Yu.;Hengesbach, Martin;Nienhaus, G. Ulrich
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Nienhaus, G. Ulrich
影响因子：
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作者：
Oyelere, AK;Kardon, JR;Strobel, SA
通讯作者：
Strobel, SA
影响因子：
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作者：
Scott, William G.
通讯作者：
Scott, William G.
影响因子：
15
作者：
Nierth, Alexander;Kobitski, Andrei Yu.;Jaeschke, Andres
通讯作者：
Jaeschke, Andres