Kinetic Isotope Effects and Stereochemical Studies on a Ribonuclease Model: Hydrolysis Reactions of Uridine 3′-Nitrophenyl Phosphate

Hengge, Alvan C.; Bruzik, Karol S.; Tobin, Aleksandra E.; Cleland, W. W.; Tsai, Ming‐Daw

doi:10.1006/bioo.2000.1170

Cited by 13 publications

(19 citation statements)

References 18 publications

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“…This result corresponds well to previous measurements by Cleland and colleagues for uridine-3′- m -nitrobenzyl phosphate (p K a 14.9) ( 18 k LG = 1.027)[23] and the large β LG observed for alkyl leaving groups [12]. The 18 k LG for uridine-3′- p -nitrophenyl phosphate (p K a 7.4) is small ( 18 k LG = 1.006)[24] suggesting an early TS along the 5′O-P bond cleavage coordinate consistent with smaller β LG for aryl leaving groups.…”

Section: Transition States Of Solution Rna 2′-o-transphosphorylationsupporting

confidence: 90%

Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2′-O-transphosphorylation

Kellerman

York

Piccirilli

et al. 2014

Current Opinion in Chemical Biology

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Although there have been great strides in defining the mechanisms of RNA strand cleavage by 2′-O-transphosphorylation, long-standing questions remain. How do different catalytic modes such as acid/base and metal ion catalysis influence transition state charge distribution? Does the large rate enhancement characteristic of biological catalysis result in different transition states relative to solution reactions? Answering these questions is important for understanding biological catalysis in general, and revealing principles for designing small molecule inhibitors. Recent application of linear free energy relationships and kinetic isotope effects together with multi-scale computational simulations are providing tentative answers to these questions for this fundamentally important class of phosphoryl transfer reactions.

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Section: Transition States Of Solution Rna 2′-o-transphosphorylationsupporting

confidence: 90%

Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2′-O-transphosphorylation

Kellerman

York

Piccirilli

et al. 2014

Current Opinion in Chemical Biology

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“…By comparison, the magnitude of the leaving group KIE for 2'- O -transphosphorylation of uridine-3'- p -nitrophenyl phosphate is small46; 49 ( ca . 18 k LG = 1.006) consistent with an early TS as observed for solvent hydrolysis of phosphodiesters with good leaving groups10.…”

Section: Discussionmentioning

confidence: 98%

“…A great advantage in interpreting KIEs for reactions involving RNA is the extensive experimental and conceptual framework derived from analyses of the KIEs of phosphodiester reactions9; 10; 11; 14; 16; 46; 47; 48; 49. Extensive studies of nucleophile, leaving group and non-bridging oxygen KIEs by Cleland and by Hengge demonstrate that these effects fall in the range of 0.93 – 1.07 ( k 16 / k 18 ) and provide important information on extents of bond formation and cleavage6; 11.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Isotope Effects for RNA Cleavage by 2′-O- Transphosphorylation: Nucleophilic Activation by Specific Base

et al. 2010

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To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary 18 O kinetic isotope effects and solvent D 2 O isotope effects were measured to probe the mechanism of base-catalyzed 2'-O-transphosphorylation of the RNA dinucleotide 5'-UpG-3'. The observed 18 O KIEs for the nucleophilic 2'-O and in the 5'-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus-oxygen bond fission to the leaving group ( 18 k LG = 1.034 ± 0.004) and phosphorous-nucleophile bond formation ( 18 k NUC = 0.984 ± 0.004). A breakpoint in the pH dependence of the 2'-Otransphosphorylation rate to a pH independent phase above pH 13 has been attributed to the pK a of the 2'-OH nucleophile. A smaller nucleophile KIE is observed at pH 12 ( 18 k NUC = 0.995 ± 0.004) that is interpreted as the combined effect of the equilibrium isotope effect (~1.02) on deprotonation of the 2′-hydroxyl nucleophile and the intrinsic KIE on the nucleophilic addition step (ca. 0.981). An alternative mechanism in which the hydroxide ion acts as a general base is considered unlikely given the lack of a solvent deuterium isotope effect above the breakpoint in the pH versus rate profile. These results represent the first direct analysis of the transition state for RNA strand cleavage. The primary 18 O KIE results and the lack of a kinetic solvent deuterium isotope effect together provide strong evidence for a late transition state and 2'-O nucleophile activation by specific base catalysis.

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“…50 Overall, the values of 18 k lg and 15 k for both the acid-catalyzed and general base-catalyzed reactions of this compound show no large-scale deviation from that measured for intermolecular acid-and basecatalyzed reactions of other model phosphodiesters. 35,48 Thus, the intramolecular nature of the reaction does not by itself have a large effect on reaction mechanism or transition state structure.…”

Section: Transition States Involving Intramolecular Attack By An Adjamentioning

confidence: 67%

“…Acid catalysis proceeds via nucleophilic attack by the adjacent 2ЈOH on the protonated neutral diester. 50 In contrast, catalysis by general bases results in partial deprotonation of the attacking 2ЈOH, while during specific base catalysis the nucleophile is the fully deprotonated alkoxide. 38 However, the degree of protonation, its effect on mechanism, as well as the charge distribution in the transition state are not well understood.…”

Section: Transition States Involving Intramolecular Attack By An Adjamentioning

confidence: 99%

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

2003

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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.

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Kinetic Isotope Effects and Stereochemical Studies on a Ribonuclease Model: Hydrolysis Reactions of Uridine 3′-Nitrophenyl Phosphate

Cited by 13 publications

References 18 publications

Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2′-O-transphosphorylation

Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2′-O-transphosphorylation

Kinetic Isotope Effects for RNA Cleavage by 2′-O- Transphosphorylation: Nucleophilic Activation by Specific Base

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

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