Specificity of aminoacyl transfer ribonucleic acid synthetases from Escherichia coli K12

Kondo, Masatoshi; Woese, Carl R.

doi:10.1021/bi00838a040

Cited by 19 publications

(7 citation statements)

References 7 publications

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“…with E. coli K-12 tRNA and the IRS purified from PB154. Inhibition of ile-tRNA formation by valine and thiaisoleucine has been reported for E. coli K-12 IRS (8,12). Figure 4 is a reciprocal plot of enzyme activity (ile-tRNA formation) as a function of ATP concentration, which shows that the affinity of the mutant enzyme for ATP is about 15-fold lower than that of the wild-type enzyme.…”

Section: Methodsmentioning

confidence: 72%

Biochemical Characterization of a Mutant Isoleucyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli K-12

Treiber

Iaccarino

1971

J Bacteriol

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Isoleucyl-transfer ribonucleic acid (tRNA) synthetase [ l -isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5; IRS] was partially purified from Escherichia coli K-12 and from an ileS mutant that appears to be altered in IRS. The half-life of wild-type IRS, incubated at 60.5 C, is 69 min, whereas that of mutant IRS is 8 min. Mutant IRS shows about a 100-fold lower affinity than wild-type IRS for isoleucine, dl -valine, thiaisoleucine, and O-methyl- dl -threonine, both in the pyrophosphate exchange assay and in the assay of isoleucyl-tRNA formation. The affinity of the mutant enzyme for adenosine triphosphate in the assay of isoleucyl-tRNA formation is 15-fold lower than that of the wild-type enzyme. The affinity of mutant IRS for tRNA is not changed as compared with wild-type IRS. These data show that mutant IRS has an altered structure and clearly confirm that ileS is the structural gene for IRS.

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Section: Methodsmentioning

confidence: 72%

Biochemical Characterization of a Mutant Isoleucyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli K-12

Treiber

Iaccarino

1971

J Bacteriol

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“…The activity in the aminoacylation reaction was affected much more than was the activity in the ATP-PP1 exchange reaction (Table 3). This may result partially from the known activation of leucine by isoleucyl-tRNA synthetase (21).…”

Section: Resultsmentioning

confidence: 99%

Isolation and Partial Characterization of Temperature-Sensitive Escherichia coli Mutants with Altered Leucyl- and Seryl-Transfer Ribonucleic Acid Synthetases

et al. 1971

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Two temperature-sensitive mutants of Escherichia coli have been found in which the conditional growth is a result of a thermosensitive leucyl-transfer ribonucleic acid (tRNA) synthetase and seryl-tRNA synthetase, respectively. The corresponding genetic loci, leuS and serS, cotransduce with lip and serC, respectively. As a result of the mutationally altered leucyl-tRNA synthetase, some leucine-, valine-, and isoleucine-forming enzymes were derepressed. Thus, leucyl-tRNA synthetase is involved in the repression of the enzymes needed for the synthesis of branched-chain amino acids.on July 16, 2020 by guest

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“…Such a positive mechanism has been demonstrated for the isoleucyl-tRNA synthetase [8] and also for leucyl-tRNA synthetase from E. coli [2], and strongly suggest that some information for the correct aminoacylation of tRNA may be supported by specific transconformations of the synthetase itself during the catalytic cycle.…”

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confidence: 76%

Isoleucyl-Transfer Ribonucleic Acid Synthetase from Bacillus stearothermophilus. 1. Properties of the Enzyme

Charlier

Grosjean²

1972

Eur J Biochem

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I n this paper we report on the properties of the pursed isoleucyl-tRNA synthetase from Bacillus stearothermophilus. The enzyme which was purified approximately 500-fold, has a molecular weight of about 110000.The effect of temperature on the activation and transfer activities has been studied. The thermostable enzyme shows an optimal activity a t 65 "C for the isoleucine-dependent ATP-PPI exchange reaction and a t 45 "C for the formation of isoleucyl-tRNA. Attempts to heat inactivate the amino-acid-transfer activity selectively, i.e. without loss of the activation reaction, were unsuccessful. The protection of the enzyme against heat inactivation in presence of tRNA makes it possible to study the binding of this molecule to the protein.An association constant of 0.11 nM-l was determined a t 70.5 "C. Formation of this tRNA enzyme complex was also demonstrated by binding onto nitrocellulose filters. By this technique one tRNA site per enzyme molecule was detected and an affinity constant of 0.4 nM-1 measured. The same results were obtained either with tRNA from Escherichia coli or B. steurothermophilus, showing close resemblance of the two nucleic acids. Periodate-oxidation of the 3'-terminal adenosine of the tRNA does not perceptibly affect the binding or the degree of protection of the enzyme against heat inactivation. These effects are still strictly dependent on magnesium ions and a clearcut requirement of these ions for the formation and/or stabilization of the tRNA * enzyme complex has been demonstrated.The intermediate isoleucine * AMP * enzyme complex with a molar ratio of 1 : 1 : 1 was isolated by molecular sieving and by chromatography on DEAE-cellulose. Mg2+ ions are required for the formation of the complex but are not necessary to stabilize the isoleucyl-adenylate once this is bound to the enzyme. The transfer of activated isoleucine to tRNA again requires magnesium ions and must probably be related to the formation of a tRNA -enzyme complex.Part only of the activated isoleucine is transferred to tRNA; this results essentially from the breakdown of a fraction of the isoleucine. AMP-enzyme complex, induced by tRNA and Mg2+ ions, acting together. Periodate-oxidized tRNA of B. stearothermophilus or of E. coli in presence of Mg2+ ions completely releases isoleucine from the complex.The major purpose of our investigation was to study the structural basis for the great specificity of the aminoacyl-tRNA synthetases. These enzymes select a single amino acid among 20 species, activate it by formation of an enzyme-bound aminoacyladenylate and subsequently transfer it to one, or a few, amino-acid-specific transfer RNAs.The specificity of the second step is higher than that of the activation step [1-31. Misrecognition between the enzyme and tRNA has been observed Enzymes. Isoleucyl-tRNA synthetase or L-isoleucine

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Specificity of aminoacyl transfer ribonucleic acid synthetases from Escherichia coli K12

Cited by 19 publications

References 7 publications

Biochemical Characterization of a Mutant Isoleucyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli K-12

Biochemical Characterization of a Mutant Isoleucyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli K-12

Isolation and Partial Characterization of Temperature-Sensitive Escherichia coli Mutants with Altered Leucyl- and Seryl-Transfer Ribonucleic Acid Synthetases

Isoleucyl-Transfer Ribonucleic Acid Synthetase from Bacillus stearothermophilus. 1. Properties of the Enzyme

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