Understanding the transition states of phosphodiester bond cleavage: Insights from heavy atom isotope effects

Abstract

The nucleotides of DNA and RNA are joined by phosphodiester linkages whose synthesis and hydrolysis are catalyzed by numerous essential enzymes. Two prominent mechanisms have been proposed for RNA and protein enzyme catalyzed cleavage of phosphodiester bonds in RNA: (a) intramolecular nucleophilic attack by the 2'-hydroxyl group adjacent to the reactive phosphate; and (b) intermolecular nucleophilic attack by hydroxide, or other oxyanion. The general features of these two mechanisms have been established by physical organic chemical analyses; however, a more detailed understanding of the transition states of these reactions is emerging from recent kinetic isotope effect (KIE) studies. The recent data show interesting differences between the chemical mechanisms and transition state structures of the inter- and intramolecular reactions, as well as provide information on the impact of metal ion, acid, and base catalysis on these mechanisms. Importantly, recent nonenzymatic model studies show that interactions with divalent metal ions, an important feature of many phosphodiesterase active sites, can influence both the mechanism and transition state structure of nonenzymatic phosphodiester cleavage. Such detailed investigations are important because they mimic catalytic strategies employed by both RNA and protein phosphodiesterases, and so set the stage for explorations of enzyme-catalyzed transition states. Application of KIE analyses for this class of enzymes is just beginning, and several important technical challenges remain to be overcome. Nonetheless, such studies hold great promise since they will provide novel insights into the role of metal ions and other active site interactions.

Conformational change in the Bacillus subtilis RNase P holoenzyme–pre-tRNA complex enhances substrate affinity and limits cleavage rate

“…We attribute these differences to the lower experimental temperature and addition of the fluorescein modification in our current study. However, the most significant result of this study is that the observed pK a is much lower than the ionization of a metal-hydroxide, the suggested nucleophile in the reaction Cassano et al 2004).…”

“…Comparative sequence analysis, site-directed mutagenesis, and phosphorothioate-substitution experiments suggest that the highly conserved helix P4 is critical for catalytic activity, but the exact location of functional groups that comprise the ribozyme active site remains undefined (Waugh and Pace 1993;Haas et al 1994;Pace and Brown 1995;Christian et al 2000;Christian et al 2002;Crary et al 2002). RNase P is proposed to catalyze the phosphodiester bond cleavage using a metal-hydroxide as the nucleophile (Cassano et al 2004), although the kinetic and mechanistic details of this reaction are still under investigation.…”

RNA fragmentation in MALDI mass spectrometry studied by H/D-exchange: Mechanisms of general applicability to nucleic acids

“…This mechanism is an intramolecular nucleophilic attack on the phosphor atom by the adjacent 2=-hydroxyl to yield a phosphorane transition state. A similar attack is described in both enzymatic [16,17] and nonenzymatic [18] reactions of RNA hydrolysis in solution. Furthermore, the phosphorane transition state is welldocumented in the mechanism of RNases [18].…”

Theoretical Comparison ofp-Nitrophenyl Phosphate and Sulfate Hydrolysis in Aqueous Solution: Implications for Enzyme-Catalyzed Sulfuryl Transfer

“…2,33,[96][97][98] , and references cited therein), as well as countless other systems (e.g. [99][100][101][102] ).…”