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

Airas, R. Kalervo

doi:10.1111/j.1432-1033.1992.tb17441.x

Cited by 5 publications

(6 citation statements)

References 19 publications

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“…The experimental material in the present work is not sufficient nor suitable to prove the existence of the proofreading segment, but its presence does not affect any of the roles of Mg2+ or polyamines observed in this work. In the least-squares optimization of the constants the k-6c value (the conformational change to reverse direction) tends to become low (Table I), which is consistent with its role of keeping the corrected and non-corrected aminoacyl species separate from each other [21].…”

Section: Resultssupporting

confidence: 61%

“…The basic reaction model in the analysis contained the normal activation and transfer steps and a slow conformational change between them (Eqn 1). The existence of the conformational-change step was considered earlier [21] and its task is to separate the pretransfer-proofreading-corrected aminoacyl-AMP from returning to free amino acid in the presence of PP,. The experimental material in the present work is not sufficient nor suitable to prove the existence of the proofreading segment, but its presence does not affect any of the roles of Mg2+ or polyamines observed in this work.…”

Section: Resultsmentioning

confidence: 99%

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Differences in the Magnesium Dependences of the Class I and Class II Aminoacyl‐tRNA Synthetases from Escherichia coli

Airas

1996

European Journal of Biochemistry

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The magnesium dependences of the ATPJPP, exchange and tRNA aminoacylation of reactions were measured for six aminoacyl-tRNA synthetases (isoleucyl-, tyrosyl-and arginyl-tRNA synthetases from class I, and histidyl-, lysyl-and phenylalanyl-tRNA synthetases from class 11). The measured values were subjected to best-fit analyses using sum square error calculations between the data and the calculated curves in order to find the mode of participation of the Mg" and to optimize the sets of the kinetic constants.The following four dependences were observed: the class I1 synthetases require three Mgz+ for the activation reaction (including the one in MgATP), but the class I synthetases require only one Mgz+ (in MgATP) ; in class I1 synthetases both MgPP, and Mg,PP, participate in the pyrophosphorolysis of the aminoacyl adenylate. Arginyl-tRNA synthetase from class I also shows a better fit if also Mg,PP, reacts, but in the isoleucyl-and tyrosyl-tRNA synthetases only MgPP, but not Mg,PP, is used in the pyrophosphorolysis. Different synthetases have different requirements for the tRNA-bound Mg2+ and spermidine, independent of the enzyme class. 1-4 Mg" or spermidines are required in the best fit models. At the end of the reaction in all the synthetases analysed the dissociation of Mg" from the product aminoacyltRNA essentially enhances the subsequent dissociation of the aminoacyl-tRNA from the enzyme. The binding of ATP to the E . aminoacyl-tRNA complex also speeds up the dissociation of the aminoacyltRNA from most of these enzymes.

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Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Differences in the Magnesium Dependences of the Class I and Class II Aminoacyl‐tRNA Synthetases from Escherichia coli

Airas

1996

European Journal of Biochemistry

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“…In the following paper [32], the possible existence of a separate proofreading segment before the transfer reaction has been discussed. Also, this mechanism is a good fit to the results in Figs 1,2 and 4, as well as Figs 1 and 3 of the previous paper [6].…”

Section: Proofreading Segmentmentioning

confidence: 99%

Analysis of the isoleucyl‐tRNA synthetase reaction by total rate equations

Airas¹

1992

European Journal of Biochemistry

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Derivation of a steady-state rate equation for the aminoacyl-tRNA synthetases is described, and its suitability for the analysis of various details of the reaction is tested. The equation is applied to the magnesium and spermidine dependences of the isoleucyl-tRNA synthetase reaction. Earlier work The transfer reaction is rather slow, and, at least under some conditions, it participates in rate limitation. (d) A Mg2+-induced reduction in the aminoacylation rate seems to be directed to the dissociation of the aminoacyl-tRNA from the enzyme. This dissociation rate is enhanced if a Mg2+ is first dissociated from the enzyme or tRNA. An increase in the Mg2+ concentration shifts the rate limitation from the transfer reaction towards dissociation of the product.The aminoacyl-tRNA synthetase reaction contains three substrates and three products, and in a simple case the total reaction has 15 intermediates (Scheme 1). If activators such as Mg2 + and polyamines are added, the number of different intermediates rises to hundreds. Consequently, the steadystate rate equation for such a reaction is complex. A total rate equation, however, could provide a means to test the compatibility of separately measured kinetic parameters, and, in addition, it could be used to test some dependences of the enzyme system. Within the aminoacyl-tRNA synthetases, the aim is to calculate the discrimination of the incorrect amino acids.Much kinetic work has been performed on the isoleucyltRNA synthetase from Escherichia coli [l -41. Therefore, it is a good model for studies using a total rate equation, too, especially to test how well the scattered parameters function with each other. In previous work [S, 61, a rate equation for isoleucyl-tRNA synthetase has been derived, the roles of Mg2+ and spermidine in the reaction analysed. The Mg2+ and polyamine dependences were used as means to test the correctness of the mechanism and to estimate various kinetic constants. A somewhat different approach for checking the compatibility of the measured parameters has been used by Okamoto and Savageau [7,8].The present paper describes kinetic measurements made by changing the tRNA concentrations. The steady-state rate equation was improved and the constants were estimated to fit the results. The analysis led to adjustments ofthe constants, especially at the transfer reaction and thereafter. The relationship between the rates of aminoacylation of tRNA and the ATP/PPi exchange (uacyl/uexch) proved to be an efficient way to test parameters at the aminoacyl adenylation step. MATERIALS AND METHODSThe isoleucyl-tRNA synthetase from E. coli B (specific activity 1.2 ymol inin-' mg protein-') and the assay mcthods

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“…The kinetic proofreading system has been a subject of discussion and mathematical calculations . It requires either an increased pyrophosphorolysis rate of the wrong aa‐AMP (in C 65 and C 21 ) or dissociation of the wrong aa‐AMP from the enzyme and hydrolysis thereafter.…”

Section: Discussionmentioning

confidence: 99%

“…If the pretransfer proofreading occurs at segment 6, before the dissociation of Ile-tRNA from the enzyme, the connection to the post-transfer editing site could be possible through Ile-tRNA. The kinetic proofreading system has been a subject of discussion and mathematical calculations [40][41][42]. It requires either an increased pyrophosphorolysis rate of the wrong aa-AMP (in C 65 and C 21 ) or dissociation of the wrong aa-AMP from the enzyme and hydrolysis thereafter.…”

Section: Relation To the Pretransfer Proofreadingmentioning

confidence: 99%

Kinetic analysis of the isoleucyl‐tRNA synthetase mechanism: the next reaction cycle can start before the previous one ends

Airas

2017

FEBS Open Bio

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Aminoacyl‐tRNA synthetases join correct amino acids to their cognate tRNA at the start of the protein synthesis. Through the kinetic analysis, it is possible to estimate how their functional details correspond to the known structural features. Kinetic analysis of the isoleucyl‐tRNA synthetase (IleRS) from Escherichia coli was accomplished. Sixteen different steady‐state two‐ligand experiments were statistically analysed simultaneously so that the same rate equations and same rate and dissociation constants applied to all experiments. The so‐called rapid equilibrium segments procedure was used to derive the rate equations. The final best‐fit mechanism included the normal activation and transfer steps, and reorganization of the steps between them and after the transfer step. In addition, the analysis strongly suggested an additional activation step, formation of a new isoleucyl‐AMP before the isoleucyl‐tRNA was freed from the enzyme. The removal of Ile‐tRNA was possible without the formation of Ile‐AMP if both isoleucine and ATP were bound to the E‐Ile‐tRNA complex, but this route covered only 11% of the total formation of Ile‐tRNA. In addition to the Mg 2+ in MgATP or MgPP i , only two tRNA‐bound Mg 2+ were required to explain the magnesium dependence in the best‐fit mechanism. The first Mg 2+ could be present in all steps before the second activation and was obligatory in the first reorganizing step and transfer step. The second Mg 2+ was present only at the transfer step, whereas elsewhere it prevented the reaction, including the activation reactions. Chloride inhibited the IleRS reaction, while 100 m m KCl caused 50% inhibition if the ionic strength was kept constant with K‐acetate. The (tRNA) value was increased from 0.057 to 1.37 μ m when the KCl concentration was increased from 0 to 200 m m . The total rate equation helps to understand the reaction route and how the simultaneous presence of Ile‐tRNA and Ile‐AMP can cause new possibilities to proofreading mechanisms of this enzyme. Enzyme Isoleucyl‐tRNA synthetase ( EC 6.1.1.5 )

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

Cited by 5 publications

References 19 publications

Differences in the Magnesium Dependences of the Class I and Class II Aminoacyl‐tRNA Synthetases from Escherichia coli

Differences in the Magnesium Dependences of the Class I and Class II Aminoacyl‐tRNA Synthetases from Escherichia coli

Analysis of the isoleucyl‐tRNA synthetase reaction by total rate equations

Kinetic analysis of the isoleucyl‐tRNA synthetase mechanism: the next reaction cycle can start before the previous one ends

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