Heavy atom labeled nucleotides for measurement of kinetic isotope effects

Abstract

Experimental analysis of kinetic isotope effects represents an extremely powerful approach for gaining information about the transition state structure of complex reactions not available through other methodologies. Implementation of this approach to the study of nucleic acid chemistry requires the synthesis of nucleobases and nucleotides enriched for heavy isotopes at specific positions. In this review we highlight current approaches to the synthesis of nucleic acids site-specifically enriched for heavy oxygen and nitrogen and their application in heavy atom isotope effect studies.

Cleaning Up Mechanistic Debris Generated by Twister Ribozymes Using Computational RNA Enzymology

“…The RNA cleavage (2′- O -transphosphorylation) reaction catalyzed by the twister ribozyme is also catalyzed by the protein enzyme RNase A . Recently, KIEs have been measured/calculated for the uncatalyzed reaction , as well as catalyzed by RNase A ,, and in the presence of Zn 2+ ions. , These reactions proceed via a largely associative mechanism, as is typical for transesterification and hydrolysis of phosphate diesters, ,, involving nucleophilic attack of the O2′ that proceeds through a dianionic pentavalent transition state. The large inverse O2′ and large normal O5′ 1° KIEs for the uncatalyzed reaction under alkaline conditions have been interpreted to suggest a considerably late transition state characterized by an almost fully formed O2′–P bond and an almost fully broken P–O5′ bond.…”

“…The RNA cleavage (2′-O-transphosphorylation) reaction catalyzed by the twister ribozyme is also catalyzed by the protein enzyme RNase A. 34 Recently, KIEs have been measured/calculated for the uncatalyzed reaction 61,64 as well as catalyzed by RNase A 57,62,65 and in the presence of Zn 2+ ions. 63,66 These reactions proceed via a largely associative mechanism, as is typical for transesterification and hydrolysis of phosphate diesters, 57,60,67 involving nucleophilic attack of the O2′ that proceeds through a dianionic pentavalent transition state.…”

Unwinding the twister ribozyme: from structure to mechanism

“…It can be influenced by four catalytic strategies: (α) orientation of the 2′ oxygen, phosphor, and 5′ oxygen atoms for in‐line nucleophilic attack; (β) electrostatic compensation of the enhanced negative charge on the nonbridging phosphate oxygens in the transition state; (γ) general base catalysis by removing the proton from the attacking 2′‐OH nucleophile; and (δ) general acid catalysis by donating a proton to the developing negative charge on the 5′ oxygen leaving group (Figure ). Moreover, we point out that recent experimental and computational evidence suggests that phosphodiester bond cleavage is not necessarily concerted but can proceed in a stepwise manner involving ‘tight’ transition states that are asynchronous . Distinct structural features of how the RNA molds a ribozyme's active site, impact on the reaction pathway and transition states for RNA 2′‐ O ‐transphosphorylation; these features are responsible for the broad range of cleavage rates that are encountered for individual classes of ribozymes.…”

Extension of the Variational Free Energy Profile and Multistate Bennett Acceptance Ratio Methods for High-Dimensional Potential of Mean Force Profile Analysis

“…This implies that cleavage of the O5′−P bond is less advanced, as is the degree of proton transfer (O5′−H bond formation) from the general acid. While this is a fairly subtle difference, it is nonetheless significant and could be detected experimentally by measurement of linear free energy relations 105,106 or kinetic isotope effects 107,108 at primary and secondary oxygen positions, similar to those carried out recently for similar reactions in the Varkud satellite ribozyme 56 and RNase A. 109,110 Hence, the ability to analyze and construct robust multidimensional free energy surfaces is important for mechanistic studies of protein and RNA enzymes.…”