Department of Physics
Phosphoryl transfer reactions are ubiquitous in biology and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate-controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition-state structure and bonding optimally requires the use of theoretical models. In this work, we apply density-functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2′-O-transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well-studied series of phosphate and phosphorothioate mono-, di- and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non-enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate–KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of building up knowledge of mechanism through the systematic study of model systems to provide insight into more complex biological systems such as phosphoryl transfer enzymes and ribozymes.
enzyme catalysis, isotope effect, leaving–group effect, reaction mechanisms, RNA
Source Publication Title
Chemistry - A European Journal
This is the peer reviewed version of the following article: Chen, H., Giese, T. J., Huang , M., Wong , K.-Y., Harris, M. E. and York, D. M. (2014), Mechanistic Insights into RNA Transphosphorylation from Kinetic Isotope Effects and Linear Free Energy Relationships of Model Reactions. Chem. Eur. J., 20: 14336–14343. doi: 10.1002/chem.201403862, which has been published in final form at http://dx.doi.org/10.1002/chem.201403862. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Financial support to D.M.Y. provided by the National Institutes of Health (GM062248), to M.E.H. provided by the National Institute of Health (GM096000) and to K.-Y.W. provided by HK RGC (ECS-209813), NSF of China (NSFC-21303151), HKBU FRG (FRG2/12-13/037) and startup funds (38-40-088 and 40-49-495). Computational resources from the Minnesota Supercomputing Institute for Advanced Computational Research (MSI) and from the High Performance Cluster Computing Centre (HPCCC) and Office of Information Technology (ITO) at HKBU (sciblade and jiraiya) were utilized in this work. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number OCI-1053575.
Link to Publisher's Edition
Chen, Haoyuan, Timothy J. Giese, Ming Huang, Kin Yiu Wong, Michael E. Harris, and Darrin M. York. "Mechanistic insights into RNA transphosphorylation from kinetic isotope effects and linear free energy relationships of model reactions." Chemistry - A European Journal 20.44 (2014): 14336-14343.