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

Abstract

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

A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli

“…[Professor Grosjean has kindly informed us that the unit ofenzyme activity in his paper should read nmol of charged tRNA formed in 10min. In terms of the units used in the present work (gmol of charged tRNA/min) the specific activity of the enzyme of Charlier & Grosjean (1972) is 1.99 units/mg, which compares well with our value of 1.28 units/mg (Table 1) Properties of valyl-tRNA synthetases from B. stearothermophilus and E. coli The molecular weight ofthe valyl-tRNA synthetase from B. stearothermophilus was determined by using polyacrylamide-gel electrophoresis under reducing and denaturing conditions. The average of seven determinations gave the molecular weight as 110000 +4000.…”

“…Leucyl-tRNA synthetase and valyl-tRNA synthetase were separated on CM52 CM-cellulose at pH 5.0, and no leucyl-tRNA or arginyl-tRNA synthetase activity could be detected in the final valyl-tRNA synthetase preparation. Isolation of valyl-tRNA synthetase from B. stearothermophilus on a smaller scale, but with a similar overall purification factor, has been reported by Charlier & Grosjean (1972). [Professor Grosjean has kindly informed us that the unit ofenzyme activity in his paper should read nmol of charged tRNA formed in 10min.…”

“…These include leucyl-tRNA synthetase (Vanhumbeeck & Lurquin, 1969;Lurquin, 1971), Vol. 139 isoleucyl-tRNA synthetase (Charlier & Grosjean, 1972) and arginyl-tRNA synthetase (Parfait & Grosjean, 1972). In all cases, as with the valyl-tRNA synthetase reported here, the enzymes have been found to be highly thermostable.…”

Aminoacyl‐tRNA Synthetases: Some Recent Results and Achievements

“…FAVOROVA Table I1 surveys the methods used to detect, to isolate, and to study the ES complexes. In addition to the methods listed, electrophoresis (103,115,122), nonequilibrium dialysis (104), influence of the substrates on the chemical modification of synthetases (82) have been also applied. Isolation of the complexes by means of gel chromatography and gel filtration is not included in the table since data on many of them have been already compiled by Allende and Allende (123).…”

“…Isolation of the complexes by means of gel chromatography and gel filtration is not included in the table since data on many of them have been already compiled by Allende and Allende (123). In addition to their table other recent publications may also be cited here (103,104,(111)(112)(113)(116)(117)(118)(119)(120)(121).…”