Distinct Kinetic Mechanisms of the Two Classes of Aminoacyl-tRNA Synthetases

Zhang, Chunmei; Perona, John J.; Ryu, Kang Sun; Francklyn, Christopher S.; Hou, Ya‐Ming

doi:10.1016/j.jmb.2006.06.015

Cited by 106 publications

(179 citation statements)

References 52 publications

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“…Under these conditions, the transition between the first and subsequent turnovers of the enzyme is visualized, providing useful information. Notably, a comparison of the pre-steady state kinetics of Class I and Class II aaRSs indicates that the former exhibit a burst of aminoacyl-tRNA in the first turnover, while the latter do not [40,62,63]. One interpretation of these data is that the overall aminoacylation catalyzed by Class I enzymes, but not Class II enzymes, is limited by the rate of product release.…”

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 98%

“…The methodology for the rapid quench method under single turnover conditions is described in earlier work by Fersht and co-workers [60], with additional recent modifications by the authors [40,62,63]. The approach will be slightly different for different enzymes, depending on whether or not the aaRS is capable of forming the aminoacyl adenylate intermediate in the absence of tRNA.…”

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 99%

“…For GlnRS, the k obs obtained represents a composite rate constant that is the limiting rate for all steps prior to product release. An additional feature of the single turnover rapid quench assay is that, provided substrate binding is in rapid equilibrium relative to the rate of transfer, the apparent dissociation constants (K d ) for amino acid and tRNA can be determined [40,63]. Another useful variation of the single turnover rapid quench assay employs varying concentrations of the aminoacyl-tRNA product as inhibitor of the forward aminoacylation reaction.…”

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 99%

“…Another useful variation of the single turnover rapid quench assay employs varying concentrations of the aminoacyl-tRNA product as inhibitor of the forward aminoacylation reaction. For CysRS, a plot of the variation in amplitude of the first order exponential against the AA-tRNA AA concentration ( Figure 5 panels C and D) can be used to extract the K d for the aminoacyl-tRNA product [63].…”

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 99%

“…To perform these experiments, reactions are prepared featuring saturating amino acid, Mg·ATP, and tRNA, allowing the maximal rate of multiple turnover aminoacylation (equivalent to k cat ) to be obtained from the linear phase of the progress curve. Representative conditions for different systems have been described in detail [40,62,63].…”

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 99%

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Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Francklyn

First

Perona

et al. 2008

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The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.

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Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 98%

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 99%

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 99%

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 99%

Section: Rapid Quench Methods For Studying the Half Reactions Of Aminmentioning

confidence: 99%

See 3 more Smart Citations

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Francklyn

First

Perona

et al. 2008

Methods

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110

120

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Gly56 in the synthetic site of isoleucyl‐tRNA synthetase confers specificity and maintains communication with the editing site

Dulic,

Krpan,

Gruic‐Sovulj

2023

FEBS Letters

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Isoleucyl‐tRNA synthetase (IleRS) links isoleucine to cognate tRNA via the Ile‐AMP intermediate. Non‐cognate valine is often mistakenly recognized as the IleRS substrate; therefore, to maintain the accuracy of translation, IleRS hydrolyzes Val‐AMP within the synthetic site (pre‐transfer editing). As this activity is not efficient enough, Val‐tRNAIle is formed and hydrolyzed in the distant post‐transfer editing site. A strictly conserved synthetic site residue Gly56 was previously shown to safeguard Ile‐to‐Val discrimination during aminoacyl (aa)‐AMP formation. Here, we show that the Gly56Ala variant lost its specificity in pre‐transfer editing, confirming that this residue ensures the selectivity of all synthetic site reactions. Moreover, we found that the Gly56Ala mutation affects IleRS interaction with aa‐tRNA likely by disturbing tRNA‐dependent communication between the two active sites.

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Strategies for Detecting Aminoacylation and Aminoacyl‐tRNA Editing In Vitro and In Cells

Watkins,

Kavoor,

Musier‐Forsyth

2024

Israel Journal of Chemistry

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Aminoacyl‐tRNA synthetases (aaRSs) maintain translational fidelity by ensuring the formation of correct aminoacyl‐tRNA pairs. Numerous point mutations in human aaRSs have been linked to disease phenotypes. Structural studies of aaRSs from human pathogens encoding unique domains support these enzymes as potential candidates for therapeutics. Studies have shown that the identity of tRNA pools in cells changes between different cell types and under stress conditions. While traditional radioactive aminoacylation analyses can determine the effect of disease‐causing mutations on aaRS function, these assays are not amenable to drug discovery campaigns and do not take into account the variability of the intracellular tRNA pools. Here, we review modern techniques to characterize aaRS activity in vitro and in cells. The cell‐based approaches analyse the aminoacyl‐tRNA pool to observe trends in aaRS activity and fidelity. Taken together, these approaches allow high‐throughput drug screening of aaRS inhibitors and systems‐level analyses of the dynamic tRNA population under a variety of conditions and disease states.

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Distinct Kinetic Mechanisms of the Two Classes of Aminoacyl-tRNA Synthetases

Cited by 106 publications

References 52 publications

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Gly56 in the synthetic site of isoleucyl‐tRNA synthetase confers specificity and maintains communication with the editing site

Strategies for Detecting Aminoacylation and Aminoacyl‐tRNA Editing In Vitro and In Cells

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