Mechanisms of aminoacyl-tRNA synthetases: a critical consideration of recent results

Freist, Wolfgang

doi:10.1021/bi00443a001

Cited by 87 publications

(44 citation statements)

References 51 publications

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“…Aminoacyl-tRNA synthetases (aaRSs) catalyze the acylation of transfer RNAs (tRNAs) with cognate amino acids via a two-step mechanism (1)(2)(3). In the first step, an amino acid is activated with ATP to form an aminoacyl-adenylate (aa-AMP) intermediate.…”

Section: Introduction: Aminoacyl-trna Synthetase Fidelitymentioning

confidence: 99%

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Splan

Musier‐Forsyth

Boniecki

et al. 2008

Methods

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Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre-and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2′ or 3′ hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.

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Section: Introduction: Aminoacyl-trna Synthetase Fidelitymentioning

confidence: 99%

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Splan

Musier‐Forsyth

Boniecki

et al. 2008

Methods

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“…The pyrophosphatase, however, does not destroy all PP,, but a moderate concentration is maintained. The PP, concentration in Escherichia roli cells, thoroughly studied in this laboratory [3 -51, is about 0.5 mM and is not changed very much by varying the conditions.The amino acid specificity of the aminoacyl-tRNA synthetases has been described to be increased in four steps [6]. Two initial discrimination steps occur before the activation reaction, the pretransfer proofreading works at the aminoacyl adenylate step and the posttransfer proofreading at the aminoacyl-tRNA step (Eqn 1).…”

mentioning

confidence: 99%

“…The amino acid specificity of the aminoacyl-tRNA synthetases has been described to be increased in four steps [6]. Two initial discrimination steps occur before the activation reaction, the pretransfer proofreading works at the aminoacyl adenylate step and the posttransfer proofreading at the aminoacyl-tRNA step (Eqn 1).…”

mentioning

confidence: 99%

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

Airas

1992

European Journal of Biochemistry

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A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E .possible. If the E . Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E . Ile-AMp with E . and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E . l'e-AMP and the corrected *E$&Mp. If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be benefitial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.The activation of amino acids by the aminoacyl-tRNA synthetases produces PP,, which is able to bind to the aminoacyl adenylate in the reversc reaction [I]. Both the activation and the pyrophosphorolysis are rapid reactions [2] which allow an equilibrium at this step in the presence of PPi. The PPi strongly shifts the equilibrium from the aminoacyl adenylate intermediates towards the start of the total reaction. PP, is decomposed by the inorganic pyrophosphatase present in all living cells. The pyrophosphatase, however, does not destroy all PP,, but a moderate concentration is maintained. The PP, concentration in Escherichia roli cells, thoroughly studied in this laboratory [3 -51, is about 0.5 mM and is not changed very much by varying the conditions.The amino acid specificity of the aminoacyl-tRNA synthetases has been described to be increased in four steps [6]. Two initial discrimination steps occur before the activation reaction, the pretransfer proofreading works at the aminoacyl adenylate step and the posttransfer proofreading at the aminoacyl-tRNA step (Eqn 1).The effect of PP, on the aminoacyl-tRNA synthetase reaction has already been considered in two earlier papers on this work [7, 81. The present work uses the kinetic equations derived in the preceding paper [9]. The roles of PP, in the initial discriminations and pretransfer proofreading were calculated. MATERIALS AND METHODS EquationsThe rate equations were derived as suggested by Cha [lo] and as described in detail in the preceding paper [9]. The reaction scheme was divided in separate rapid equilibrium segments. Since the amino acid specificity is to be examined, the binding reaction for the amino acid makes one boundary of the segments, and the different amino acids, isoleucine and valine, have their own segments (Eqn 2).

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“…Then v = k - 3[Eo]IJ'(). The rate equations derived were used in calculating and drawing the curves corresponding to the experimental results with a computer.…”

Section: Rate Equationsmentioning

confidence: 99%

On the roles of magnesium and spermidine in the isoleucyl‐tRNA synthetase reaction

Airas

1990

European Journal of Biochemistry

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The reaction of isoleucyl-tRNA synthetase from Escherichiu coli B was analysed by deriving total steady-state rate equations for the ATPjPP, exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2 + inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine . ATP complex. Spermidine . ATP is a weaker substrate. The role of spermidine . ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.The general model for the aminoacyl-tRNA synthetase reactions contains the activation of the amino acid by ATP through formation of an enzyme-bound aminoacyl-adenylate intermediate, and thereafter transfer of the aminoacyl moiety to the tRNA [I -31. A Mg2+ can theoretically participate in the total reaction by binding to the enzyme [4, 51, ATP [6] or tRNA [7-91. It is obligatory in the activation reaction, but most of it can be replaced by spermidine or spermine in the transfer step or total reaction [4, 10, 111. Many of the characteristics of the Mg2+ dependence of the isoleucyl-tRNA synthetase reaction can be explained by a model where MgATP always is one of the substrates, and a second Mg2+ is required in the tRNA, and the second Mg2 + can be replaced by spermidine [8].In some recent papers we have developed total steady-state rate equations to describe various properties of the aminoacyltRNA synthetase reactions [12 -141. This approach inevitably has limitations due to the great complexity of the total reaction and due to the unknown values of most of the kinetic constants. However it has proved to be useful as a supplement to the earlier kinetic treatments. In the present paper, some features of the isoleucyl-tRNA synthetase reaction were analysed by expanding the total rate equation to the Mg2+ and spermidine effects on the aminoacylation of tRNA and the ATP/PPi exchange reaction. The results suggest a participation of two Mg2+ in the activation reaction. One of them can be replaced by a polyamine. In the best chosen mechanism the Mg2+ in ATP can be replaced by spermidine and the other, enzyme-bound Mg2 + is required only in the absence of tRNA.

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Mechanisms of aminoacyl-tRNA synthetases: a critical consideration of recent results

Cited by 87 publications

References 51 publications

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl‐tRNA synthetase from Escherichia coli

On the roles of magnesium and spermidine in the isoleucyl‐tRNA synthetase reaction

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