Arginyl Transfer Ribonucleic Acid Synthetase Research Articles

Arginyl-transfer ribonucleic acid synthetase of Bacillus stearothermophilus. Purification and kinetic analysis.

Arginyl-tRNA synthetase from Bacillus stearothermophilus (NCA 1518) has been purified 880-fold to apparent homogeneity as demonstrated by electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight is 59 000 as confirmed by Sephadex G-100 and by sucrose gradient ultracentrifugation. The enzyme is monomeric, no subunits were detected. Its cognate tRNA induces an apparent increase in molecular weight suggesting the dimerisation of the enzyme. Nevertheless, it is not obvious that the enzyme dimer forms prior to the aminoacylation reaction catalysed by the enzyme. An ATPase activity was found associated to the synthetase but can be neglected because the ATP consumption is too low for hampering the arginyl-tRNA synthetase activity. The order of addition of substrates and release of products has been studied by measurements of initial velocity, product inhibition and dead-end inhibition. The nature of the kinetic patterns indicates that the aminoacylation reaction conforms to the classical concept of the mechanism which includes the formation of an enzyme-bound aminoacyl-adenylate as an intermediate in the first step followed by transfer of the amino acid to tRNA. The first partial reaction, measured by the ATP-32PPi exchange or AMP synthesis in the presence of ATP and arginine, requires tRNA, which is consistent with the model in which tRNAArg is an activator of the arginyladenylate synthesis.

Adenosine triphosphate consumption by bacterial arginyl-transfer ribonucleic acid synthetases

ATP consumption by arginyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus has been investigated by the firefly luciferin--luciferase assay. Arginyl-tRNA synthetase from E. coli utilizes ATP only for aminocylation of tRNA with a 1:1 stoicheiometry. In contrast, we have shown an adenosine triphosphatase activity of arginyl-tRNA synthetase from B. stearothermophilus in the absence of tRNAArg. Dowex chromatography revealed the formation of ADP by the thermophile enzyme; under aminoacylation conditions, AMP was also formed in amounts stoicheiometric with arginyl-tRNA formation.

Control of arginine biosynthesis in Escherichia coli: inhibition of arginyl-transfer ribonucleic acid synthetase activity.

In this study, we have extended our earlier observations indicating in vitro inhibition of arginyl-transfer ribonucleic acid synthetase (EC 6.1.13, arginine: soluble ribonucleic acid ligase, adenosine monophosphate) activity by the arginine biosynthetic precursors ornithine, citrulline, and argininosuccinate. Furthermore, we report evidence which suggest that this enzyme activity is inhibited by these arginine precursors in vivo and that this inhibition of activity results in a derepression of arginine biosynthesis.

Control of arginine biosynthesis in Escherichia coli: characterization of arginyl-transfer ribonucleic acid synthetase mutants.

The arginyl-transfer ribonucleic acid (Arg-tRNA) synthetase (EC 6.1.1.13, arginine: RNA ligase adenosine monophosphate) mutants, exhibiting nonrepressible synthesis of arginine by exogenous arginine, were employed in studies of several biochemical properties. Two of these mutants possessed Arg-tRNA synthetases with a reduced affinity for arginine, and this enzyme of another mutant had a reduced affinity for arginine-tRNA (tRNA(arg)). The mutant possessing an Arg-tRNA synthetase with an altered K(m) for tRNA(arg) was found to have reduced in vivo aminoacylation of two of the five isoaccepting species of tRNA(arg) and complete absence of aminoacylation of one of the isoaccepting species.

Control of arginine biosynthesis in Escherichia coli: role of arginyl-transfer ribonucleic acid synthetase in repression.

The physiological role of arginyl-transfer ribonucleic acid (Arg-tRNA) synthetase (E.C. 6.1.1.13, arginine: RNA ligase adenosine monophosphate) in repression of arginine biosynthetic enzymes was examined. Mutants with nonrepressible synthesis of arginine biosynthetic enzymes were isolated from various strains of Escherichia coli by resistance to growth inhibition by canavanine, an arginine analogue. These mutants possessed reduced Arg-tRNA synthetase activities which were qualitatively different from the synthetase activity of the wild type. The mutant enzymes exhibited turnover in vivo and were less stable in vitro than the wild type at both 4 C and 40 C; they possessed different affinities for both arginine and canavanine as measured by the three common assay systems for aminoacyl-tRNA synthetases. Furthermore, in one case it was shown that (i) the mutant possesed unaltered uptake of arginine, and (ii) that the mutant possessed diminished ability to incorporate canavanine into proteins and to attach canavanine to tRNA. These observations suggested that the mutation to canavanine resistance involved a structural change in Arg-tRNA synthetase. Likewise, the results of genetic experiments suggested that the mutants differed from the wild-type strain at only one locus, and that this lies in the region of the chromosomes that includes a structural gene for Arg-tRNA synthetase. It appears that Arg-tRNA synthetase may be involved in some way in repression by arginine of its own biosynthetic enzymes.

Evidence for the existence of two arginyl-transfer ribonucleic acid synthetase activities in Escherichia coli.

Two arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: ribonucleic acid ligase adenosine monophosphate) activities were found in extracts of Escherichia coli strains AB1132 and NP2. The two arginyl-tRNA synthetase activities in extracts of strain AB1132 were found to be separable by diethylaminoethyl-cellulose column chromatography, Sephadex column fractionation, and by sucrose density gradient centrifugation. In addition, in the standard assay using extracts of strain AB1132 there were two pH optima for arginyl-tRNA synthetase activity. Furthermore, when arginyl-tRNA synthetase of strain NP2 was fractionated by hydroxylapatite column chromatography, two activities were observed which were similar to those of strain AB1132.

A random sequential mechanism for arginyl transfer ribonucleic acid synthetase of Escherichia coli.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRandom sequential mechanism for arginyl transfer ribonucleic acid synthetase of Escherichia coliTakis S. Papas and Alan PeterkofskyCite this: Biochemistry 1972, 11, 24, 4602–4608Publication Date (Print):November 1, 1972Publication History Published online1 May 2002Published inissue 1 November 1972https://doi.org/10.1021/bi00774a029RIGHTS & PERMISSIONSArticle Views39Altmetric-Citations44LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (828 KB) Get e-Alerts Get e-Alerts

Repression of enzymes of arginine biosynthesis by L-canavanine in arginyl-transfer ribonucleic acid synthetase mutants of Escherichia coli.

We show that the arginine analogue, l-canavanine, repressed the accumulation of translatable messenger ribonucleic acid (RNA) for three arginine biosynthetic enzymes in Escherichia coli. The method used to determine the level of translatable messenger RNA depended upon measurement of a burst of enzyme synthesis as described previously. E. coli strains with defective arginyltransfer ribonucleic acid (tRNA) synthetase (argS mutants) were insensitive to canavanine repression. When deprived of leucine, a leu argS strain regained normal sensitivity to canavanine repression. The level of in vivo canavanyl-tRNA(arg) was determined for a normal strain and an argS mutant. After 20 min of growth with canavanine only 9% of tRNA(arg) from the argS strain was protected from periodate oxidation, while 42% of the tRNA(arg) from an argS(+) strain was charged. When deprived of leucine, leu argS or leu argS(+) strains grown with canavanine contained more than 60% charged tRNA(arg). Reverse phase column chromatography of periodate-oxidized tRNA from canavanine-grown argS and argS(+) strains showed no preferential charging of any isoaccepting species of tRNA(arg). Therefore, we failed to detect a specific arginyl-tRNA species that might be involved in repression by canavanine. However, the data suggest that canavanine repression of the arginine pathway occurs only when high levels of canavanyl-tRNA are present, and thus support the notion that arginyl-tRNA synthetase plays a role in generating a repression signal.

Arginyl-transfer ribonucleic-acid synthetase from Bacillus stearothermophilus. Purification, properties and mechanism of action.

Arginyl‐tRNA synthetase from Bacillus stearothermophilus (NCA 1518) has been purified approximately 450‐fold to 90%, homogeneity. The enzyme has a molecular weight of 78000 as estimated by disc electrophoresis in denaturing conditions (dodecylsulfate). No subunits were revealed in these conditions. The Km values for arginine, ATP and tRNA have been determined. Like the arginyl‐tRNA synthetase from other organisms, this one from B. stearothermophilus requires tRNA to catalyse an arginine‐dependent ATP/[32P]PPi exchange reaction. Periodatetreated tRNA does not induce this exchange reaction. Aminoacylation is not completely inhibited by 2 mM pyrophosphate, indicating that the exchange reaction may be explained by the reversal of the overall aminioacylation reaction.Different methods tested failed to reveal any arginyl‐adenylate intermediary. By gel filtration, however, enzyme‐bound arginine could be isolated from a system containing all the substrates of the enzyme.The binding of arginine and ATP to arginyl‐tRNA synthetase has been studied by equilibrium partition and shows that arginine is bound only after the binding of tRNA and ATP.The binding of ATP does not depend on the presence of other substrates in the reaction mixture. One binding site for arginine and one for ATP are found per molecule of active enzyme. Periodate‐oxidized tRNA and phenoxyacetylarginyl‐tRNA did not induce the binding of arginine to the enzyme. Intact tRNA from other species which could be aminoacylated by arginyl‐tRNA synthetase from B. stearothermophilus, also induced the binding of arginine to the enzyme. Nucleotides other than ATP were not effective in the binding of arginine.It is concluded that the mechanism of action of this enzyme is a concerted one, as proposed by Loftfield and Eigner, in which arginine is bound after tRNA and ATP.

Inhibition of Arginyl-Transfer Ribonucleic Acid Synthetase Activity of Escherichia coli by Arginine Biosynthetic Precursors

The arginine biosynthetic precursors, ornithine, citrulline, and argininosuccinate, inhibit arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: soluble RNA ligase, adenosine monophosphate) activity in the in vitro attachment assay system. Ornithine is the most potent, argininosuccinate is next, and citrulline is least effective. The implications of these results are discussed in relation to arginyl-tRNA synthetase activity and the level of the arginine biosynthetic enzymes during conditions of restricted and unrestricted supply of arginine to cells.

Differences in the Thermal Inactivation Properties of Lysyl and Arginyl Transfer Ribonucleic Acid Synthetases of Bakers' Yeast

Abstract An investigation was made of the thermal inactivation of lysyl-tRNA synthetase (l-lysine:tRNA ligase (AMP), EC 6.1.1.6) 400-fold purified from bakers' yeast. During heat inactivation at pH 7.2, 45°, the enzyme was stabilized by the presence of l-lysine or by ATP and MgCl2. The concentration of l-lysine giving half-maximal protection of the enzyme was decreased by the presence of ATP and MgCl2, or tRNA and MgCl2. Similarly, polyuridylate and polycytidylate enhanded the protection of the synthetase by l-lysine. No stabilization was given by any polynucleotide in the absence of l-lysine. Adenosine 5'-phosphate, adenosine 2'(3')-monophosphate, adenosine, 5-hydroxy-dl-lysine, and ethylene glycol each gave some stabilization of the enzyme in certain conditions. In a comparative study it was found that partially purified preparations of arginyl-tRNA synthetase (l-arginine:tRNA ligase (AMP), EC 6.1.1) from bakers' yeast showed a different pattern of susceptibility to heat from that shown by the lysine enzyme. Low concentrations of l-arginine were less effective than tRNA and MgCl2 in stabilization of arginyl-tRNA synthetase from thermal inactivation at 50°. The greatest protection was observed in the presence of l-arginine, tRNA, and MgCl2, whereas l-arginine, ATP, and MgCl2 gave lower protection. The characteristics of heat inactivation of the yeast aminoacyl-tRNA synthetases specific for lysine and arginine could be interpreted in terms of substrate-induced conformational changes in the enzyme proteins.

Expression of the arginine regulon of Escherichia coli W: evidence for a second regulatory gene.

Effect of the M (modifier) gene of Escherichia coli W on the expression of wild-type structural genes of four arginine biosynthetic enzymes was studied by examining enzyme activity in cell-free extracts of cultures grown in minimal medium and medium containing arginine. The mutant M gene was originally identified as causing arginine-induced synthesis of acetylornithine delta-transaminase in a strain deficient for the enzyme. The strains used in this study received the mutant M gene by recombination. Noncoordinate repression has been demonstrated for two more enzymes of the arginine regulon of E. coli W and the M(-) gene increases the degree of noncoordinate repression for the regulon. Mutation of the M gene results in altered regulation of acetylornithine delta-transaminase, ornithine transcarbamylase, and acetylornithinase. In addition, a decreased growth rate is observed. It is proposed that the M gene is a regulatory gene. A model is presented to explain the data which involves changes in operator-repressor affinity for the structural genes and possibly for the gene controlling arginyl transfer ribonucleic acid synthetase.

The Biochemical Characterization of Two Mutant Arginyl Transfer Ribonucleic Acid Synthetases from Escherichia coli K-12

Abstract The arginyl transfer RNA synthetase from Escherichia coli K-12, wild-type, and two arginyl-tRNA synthetase mutant strains, MA 5003 and MA 5005, was purified approximately 200, 140, and 180 times, respectively. Various parameters of these synthetases were measured with the use of the esterification of arginine to transfer RNA as the assay. The mutant enzymes were found to have certain altered properties in common. Both mutant synthetases displayed an increased thermal sensitivity when compared to the wild-type enzyme. The pH optimum for all three enzymes was 8.0, but the activity of the mutant synthetases decreased more rapidly than the activity of the wild-type enzyme as the pH was lowered from 8.0 to 5.5. The Vmax of the mutant synthetases was lower than that of the wild-type enzyme when parameters as the Km values for arginine, ATP, tRNA, and dATP were measured. What differentiated the two mutant synthetases was the observation that the enzyme from strain MA 5003 displayed a 10- to 15-fold increase in Km for arginine, but less than a 2-fold increase in Km for ATP, whereas the arginyl-tRNA synthetase from strain MA 5005 had a 10-fold increase in Km for ATP, but less than a 2-fold increase in Km for arginine compared to the wild-type enzyme. It appears that the mutation in each mutant enzyme independently affects the Km for one substrate, arginine or ATP. When dATP was substituted for ATP, no difference in Km was observed between the wild-type enzyme and the mutant enzyme from strain MA 5005. Lastly the Km for tRNA was the same for all three enzymes.

The Activation of Arginyl Transfer Ribonucleic Acid Synthetase by Transfer Ribonucleic Acid

Abstract The arginine-activating enzyme of Escherichia coli requires transfer ribonucleic acid (tRNA) for catalysis of the ATP-pyrophosphate exchange reaction. Only the specific arginine-accepting tRNA is effective, and the terminal adenylic acid residue is essential for the reaction. A previously described isotope-trapping experiment led to the suggestion that an arginyl-tRNA ester was not formed during the pyrophosphate exchange. The specific tRNA has now been shown to be required for reaction of this enzyme system with hydroxylamine and for the enzymatic pyrophosphorolysis of arginyl adenylate.