Characterization of Isoleucyl-tRNA Synthetase from Staphylococcus aureus

Pope, Andrew J.; Lapointe, Jacques; Mensah, Lucy; Benson, Neil; Brown, Murray J. B.; Moore, Keith J. M.

doi:10.1074/jbc.273.48.31680

Cited by 34 publications

(27 citation statements)

References 27 publications

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“…Moreover, characterization of the IleRS reaction cycle (12) showed that binding of Ile and ATP is thermodynamically linked, making this factor more important. Rather than seeking an analytical solution to this problem, we approached it through numeric simulation techniques, using the reaction state model that we had developed for binding and activation of substrates by IleRS (12). Simulations of steadystate inhibition predicted that compounds which competed with both Ile and ATP would yield linear competitive inhibition kinetics (i.e.…”

Section: Resultsmentioning

confidence: 99%

“…We therefore also characterized the kinetics of substrate binding and activation in some detail. This work is reported in an accompanying paper (12) and the results summarized in the scheme given in Table I. The purpose of this was 3-fold.…”

mentioning

confidence: 99%

“…Our ultimate goal is to investigate how a more rational approach could be taken to inhibitor design, particularly with respect to overcoming resistance. We have characterized in detail the kinetics and mechanism of substrate binding and catalysis by S. aureus IleRS (12) and, in the present paper, inhibition by PS-A analogues. Understanding the detailed structure-activity relationships for this class of inhibitor has subsequently led to the development of even more potent compounds.…”

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confidence: 99%

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Characterization of Isoleucyl-tRNA Synthetase from Staphylococcus aureus

Pope¹,

Moore²,

McVey³

et al. 1998

Journal of Biological Chemistry

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Section: Resultsmentioning

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Characterization of Isoleucyl-tRNA Synthetase from Staphylococcus aureus

Pope¹,

Moore²,

McVey³

et al. 1998

Journal of Biological Chemistry

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“…Design-For simplicity, the following analysis is based on the TyrRS system; comparative analyses on the isoleucyl-and threonyl-tRNA synthetases have been reported [43,74]. Rate and dissociation constants refer to the reaction mechanism shown in Figure 8.…”

Section: Methodsmentioning

confidence: 99%

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Francklyn

First

Perona

et al. 2008

Methods

106

120

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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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“…Most pre-steady-state kinetic studies of tRNA synthetases have addressed the adenylate synthesis step, which proceeds independently of tRNA for most of the enzymes (30)(31)(32)(33)(34). However, early studies of MetRS, PheRS, TyrRS, ArgRS, and TrpRS used chemical-quench flow to examine aminoacylation with 14 C-labeled amino acid (35)(36)(37)(38)(39).…”

Section: Assay For Steady-state and Single-turnover Aminoacylation Kimentioning

confidence: 99%

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Uter

Perona

2004

Proc. Natl. Acad. Sci. U.S.A.

107

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Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k cat. Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA Gln . Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k chem) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine Kd is significantly higher than the steady-state K m value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k chem by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 Å distant. By contrast, mutation of the adjacent G36 has very small effects on k chem and Kd for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex.R ecognition between proteins and RNA generally occurs by an induced-fit mechanism, in which the protein, the RNA, or both undergo conformational changes en route to the final bound complex (1, 2). Although induced fit necessarily incurs an entropic penalty compared with a preformed and rigid interaction, the built-in flexibility nonetheless may help to increase the kinetic association rate, thus lowering the free-energy barrier for complex formation. In enzymatic reactions, it is also established that induced fit can provide a means to increase substrate specificity: Noncognate substrates may induce partial or incorrect rearrangements, leading to misalignment of reactive moieties along the pathway to the transition state (3). Thus, enzymes that modify RNA may employ induced fit for substrate discrimination at both binding and catalytic steps of the reaction.Aminoacyl-tRNA synthetases are superb model systems for investigating the operation of induced fit in enzyme-RNA complexes. In all organisms, these enzymes maintain fidelity of the genetic code by catalyzing the synthesis of cognate aminoacyl-tRNAs for use in protein synthesis (4). This synthesis occurs by means of a two-step reaction in which the amino acid is first activated to form an aminoacyl adenylate intermediate with release of pyrophosphate (PP i ). In the second step, the nucleophilic oxygen from the 2Ј-OH or 3Ј-OH group on the 3Ј-terminal tRNA ribose sugar attacks the mixed anhydride interm...

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Characterization of Isoleucyl-tRNA Synthetase from Staphylococcus aureus

Cited by 34 publications

References 27 publications

Characterization of Isoleucyl-tRNA Synthetase from Staphylococcus aureus

Characterization of Isoleucyl-tRNA Synthetase from Staphylococcus aureus

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

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