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

Airas, R. Kalervo

doi:10.1111/j.1432-1033.1992.tb17440.x

Cited by 8 publications

(4 citation statements)

References 35 publications

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“…The specific activity of purified tRNA11' was 0.5 nmol / A260 unit. This value is in agreement with the previously published value, 0.5 nmol/^o unit, for purified E. coli tRNA11' (Airas, 1992).…”

Section: Methodssupporting

confidence: 93%

Probing the metal binding sites of Escherichia coli isoleucyl-tRNA synthetase

Xu¹,

Trawick²,

Krudy³

et al. 1994

Biochemistry

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The metal binding properties of isoleucyl-tRNA synthetase (IleRS) from Escherichia coli were studied by in vivo substitution of the enzyme-bound metals. Purified E. coli IleRS was shown to have two tightly bound zinc atoms per active site. Cobalt- and cadmium-substituted IleRS were also found to contain two tightly bound Co2+ and Cd2+ atoms per polypeptide chain, respectively. The d-d transitions in the low energy absorption spectrum of Co(2+)-substituted IleRS were characteristic of that expected for two tetrahedrally coordinated Co2+ metals. Apo-IleRS was found to be inactive in both the aminoacylation of tRNA(Ile) and in the isoleucine-dependent ATP-pyrophosphate exchange reactions. Both Co(2+)- and Cd(2+)-substituted IleRS were found to have kcat/Km values in the isoleucine-dependent ATP-pyrophosphate exchange assay approximately 5-fold lower than the native Zn2+ enzyme. A single enzyme-bound Zn2+ or Co2+ atom per polypeptide chain could be removed by dialysis of Zn(2+)- or Co(2+)-substituted IleRS against 1,10-phenanthroline. Removal of one of the two enzyme-bound Zn2+ atoms per polypeptide chain with 1,10-phenanthroline was found to decrease (kcat/Km)Ile by approximately 130-fold. The dependence of the kinetic parameters on the identity and number of enzyme-bound metals in the isoleucine-dependent ATP-pyrophosphate exchange reaction suggests that at least one enzyme-bound metal is indirectly involved in aminoacyladenylate formation. Metal substitution or removal of one of the two enzyme-bound metals in IleRS was found to have little effect on the Km value for tRNA(Ile) or the kcat value for aminoacylation of tRNA(Ile).(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodssupporting

confidence: 93%

Probing the metal binding sites of Escherichia coli isoleucyl-tRNA synthetase

Xu¹,

Trawick²,

Krudy³

et al. 1994

Biochemistry

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“…1 shows the Mg2+ dependence of the Ile-tRNA synthetase reaction. In the earlier analysis Ile-tRNA synthetase was shown to require one Mg2+ in the ATP, one Mg" or spermidine in the enzyme and at least two Mg" or spermidines in the tRNA [12]. Statistical optimization of the reaction model led to the lowest errors if four Mg2+ or spermidines were bound to the tRNA, but the present experimental material did not require an enzyme-bound Mg2+.…”

Section: Resultsmentioning

confidence: 50%

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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“…The equations were derived as described previously [8,10]. The following steps were included: 1 The total reaction scheme was divided into 4-6 segments, depending on the mechanism to be tested.…”

Section: Equations and Best-fit Analysismentioning

confidence: 99%

“…In some previous works, I have applied the ‘rapid equilibrium segments’ procedure to derive rate equations for aminoacyl‐tRNA synthetases . These analyses included the dependences on the magnesium and polyamine concentrations and the dissociation of Mg 2+ from Ile‐tRNA before it is freed from the enzyme , the differences in the magnesium dependences between the Class I and Class II aminoacyl‐tRNA synthetases , and chloride inhibition . Due to limited experimental material, those studies did not lead to equations that could be satisfactorily used for new different experiments.…”

mentioning

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

Cited by 8 publications

References 35 publications

Probing the metal binding sites of Escherichia coli isoleucyl-tRNA synthetase

Probing the metal binding sites of Escherichia coli isoleucyl-tRNA synthetase

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

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

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