The Salk Institute, Jack H. Skirball Center for Chemical Biology and Proteomics, 10010 North Torrey Pines Road, La Jolla, California 92037, United States.
Department of Chemistry, Dana and David Dornsife College of Letters, Arts, and Sciences, University of Southern California, University Park Campus, Los Angeles, California 90089, United States.
Biochemistry. 2021 Jan 12;60(1):1-5. doi: 10.1021/acs.biochem.0c00779. Epub 2020 Dec 27.
A recently described DNA polymerase ribozyme, obtained by evolution, provides the opportunity to investigate mechanistic features of RNA catalysis using methods that previously had only been applied to DNA polymerase proteins. Insight can be gained into the transition state of the DNA polymerization reaction by studying the behavior of various β,γ-bridging substituted methylene (CXY; X, Y = H, halo, methyl) or imido (NH) dNTP analogues that differ with regard to the p of the bisphosphonate or imidodiphosphate leaving group. The apparent rate constant () of the polymerase ribozyme was determined for analogues of dGTP and dCTP that span a broad range of acidities for the leaving group, ranging from 7.8 for the CF-bisphosphonate to 11.6 for the CHCH-bisphosphonate. A Brønsted plot of log() versus p of the leaving group demonstrates linear free energy relationships (LFERs) for dihalo-, monohalo-, and non-halogen-substituted analogues of the dNTPs, with negative slopes, as has been observed for DNA polymerase proteins. The unsubstituted dNTPs have a faster catalytic rate than would be predicted from consideration of the linear free energy relationship alone, presumably due to a relatively more favorable interaction of the β,γ-bridging oxygen within the active site. Although the DNA polymerase ribozyme is considerably slower than DNA polymerase proteins, it exhibits a similar LFER fingerprint, suggesting mechanistic commonality pertaining to the buildup of negative charge in the transition state, despite the very different chemical compositions of the two catalysts.
一种最近通过进化获得的 DNA 聚合酶核酶，为使用以前仅应用于 DNA 聚合酶蛋白的方法研究 RNA 催化的机制特征提供了机会。通过研究各种β，γ-桥接取代亚甲基（CXY；X，Y = H，卤素，甲基）或亚氨基（NH）dNTP 类似物的行为，可以深入了解 DNA 聚合反应的过渡态，这些类似物在双磷酸酯或亚氨二磷酸酯离去基团的 p 方面有所不同。聚合酶核酶的表观速率常数（）是通过跨越离去基团的广泛酸度范围的 dGTP 和 dCTP 类似物确定的，从 CF-双磷酸酯的 7.8 到 CHCH-双磷酸酯的 11.6。log（）与离去基团 p 的 Brønsted 图表明，dNTP 的二卤代、单卤代和非卤代取代类似物具有线性自由能关系（LFER），斜率为负，这与 DNA 聚合酶蛋白的观察结果一致。未取代的 dNTP 的催化速率比仅考虑线性自由能关系所预测的要快，这可能是由于β，γ-桥接氧在活性部位的相互作用相对更有利。尽管 DNA 聚合酶核酶比 DNA 聚合酶蛋白慢得多，但它表现出相似的 LFER 指纹，这表明尽管两种催化剂的化学组成非常不同，但过渡态中负电荷的积累具有机制上的共同性。
