Mechanistic Insights into RNA Transphosphorylation from Kinetic Isotope Effects and Linear Free Energy Relationships of Model Reactions

Abstract

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.

Integration of kinetic isotope effect analyses to elucidate ribonuclease mechanism

“…Experiment and computation together indicate that RNase A stabilizes a product-like transition state that is very similar to the anionic solution mechanism catalyzed by specific base in solution[12, 34, 36, 60]. Nonetheless, the enzyme significantly alters leaving group bonding attributable to general acid catalysis from His119.…”

“…The substrate dinucleotide RNA molecule is selected in the first round of mass spectrometry, and the fragments of this parent ion resulting from inert gas collision are analyzed by a second round of mass spectrometry. The enhanced signal to noise allows the 16 O/ 18 O ratio to be measured with sufficient precision to allow mechanistic interpretations[12, 34, 36, 60]. The tandem MS/MS analysis of whole RNA molecules permits the KIE on the 2’O nucleophile to be measured, which is more difficult to obtain using the remote label approach.…”

VR-SCOSMO: A smooth conductor-like screening model with charge-dependent radii for modeling chemical reactions

“…4,6,[15][16][17][18][19][20] Two different types of phosphoryl reactions were used to test the phosphate hydrolysis applicability of the DFTB3+VR-SCOSMO framework to RNA-like systems, a phosphate hydrolysis reaction, and a series of phosphoryl transesterification transfer reactions with different leaving groups. These reactions have a large amount of local charge transfer, totaling a net 2e charge, which makes them ideal candidates for use with a charge-dependent model with an aim of accurately modeling biological reactions.…”

“…4,6,[15][16][17][18][19][20][21][22] These phenomena are especially apparent when studying highly charged systems such as RNA 11 as shown in Figure 1. To model these effects, simulations can be performed that include a vast buffer of explicit solvent molecules; however, such approaches are often extremely computationally demanding.…”

“…Such studies can help interpret experimental results which are pursuing a deeper understanding of the fundamental principles governing chemical activity and encompass a wide range of archetypes including, but not limited to, reactions occurring within protein environments, [1][2][3] model phosphoryl transfer reactions, [4][5][6][7][8] and studies of RNA catalysis. [9][10][11] The end goal of these studies is frequently to aid in the design of new drugs and therapeutic treatments.…”