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Results for “Methionine-tRNA Ligase”

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At least 19 recordsLinked to original sources
PubMed1976

Comparative effect of methioninyl adenylate on the growth of Salmonella typhimurium and Pseudomonas aeruginosa.

The bacteriostatic effect of methioninyl adenylate(MAMP)--a specific inhibitor of the enzyme methionyl-tRNA synthetase--was investigated on Salmonella typhimurium and Pseudomonas aeruginosa. 0.1 mM of this molecule added to the culture, inhibits the growth of S. typhimurium. The inhibition is specifically reversible by 0.1 mM L-methionine. In the same conditions even 1-2 mM MAMP has a very slight effect on the growth rate of P. aeruginosa and only during the first two generations. The same observation was made with the two other members of the fluorescens group P.fluorescens and P.putida. The growth rate of P. testosteroni with 1 mM MAMP in the medium is similar to the growth rate of P. aeruginosa but the other member of the acidovorans group P. acidovorans is much more affected by the smae concentration of the inhibitor. --P. multivorans is inhibited by MAMP like P. acidovorans but with a somewhat higher yield at the end of the culture. --MAMP has no effect on P. alcaligenes. The possible reasons for the weak bacteriostatic effect of MAMP on P. aeruginosa were investigated. It was established that the inhibitor enters the cells and is not used as a carbon and energy source. The intracellular methionine concentration in S. typhimurium and in P. aeruginosa is about the same and does not increase when bacteria are cultivated with MAMP. The MTS of the two microorganisms is inhibited by MAMP in vitro to about the same extent. Furthermore the tRNAmet from P. aeruginosa are fully acylated after 3 to 4 generations with this compound. Nevertheless MAMP elicits higher MTS activity in P. aeruginosa and in P. acidovorans after 1 h of incubation. The most striking difference between S. typhimurium and P. aeruginosa is that the intra and extracellular level of 5'phosphodiesterase which degrades MAMP is 10-20 fold higher in the second than in the first species.

Adenosine Monophosphate↗
PubMed1977

Sulphur metabolism in Paracoccus denitrificans. Purification, properties and regulation of cysteinyl-and methionyl-tRNA synthetase.

Cysteinyl- and methionyl-tRNA synthetases (EC 6.11.-) were purified 1200- and 1000-fold, respectively, from sonic extracts of Paracoccus denitrificans strain 8944, and kinetics, substrate specificity and regulatory properties were determined using the ATP-PPi exchange reaction. Both enzymes had pH optima of approx. 8 and were inhibited by sulphydryl-group reagents. Cysteinyl-tRNA synthetase catalysed L-selenocysteine- and alpha-aminobutyric acid-dependent ATP-PPi exchange and methionyl-tRNA synthetase catalysed L-homocysteine-, L-selenomethionine- and norleucine-dependent ATP-PPi exchange. Both enzymes were inhibited by O-acetylserine. Cysteinyl-tRNA synthetase activity was stimulated by methionine and methionyl-tRNA synthetase activity was stimulated by sulphide, cysteine, and cysteic acid.

Adenosine Triphosphate↗
PubMed1977

Further studies of the action of methionyl adenylate on chick embryo fibroblasts.

Methionyl adenylate (Met-AMP) inhibits protein synthesis by interacting with methionyl-tRNA synthetase. Addition of 1--3 mM inhibitor to chick embryo fibroblasts rapidly stops protein synthesis and DNA synthesis but not RNA synthesis. These effects can be reversed by renewal of the medium. The extent and reversibility of protein and DNA syntheses depend on the concentration of MetAMP in the cultures, the length of exposure and the cellular density. MetAMP is recognised by several enzymes as substrate and/or as inhibitor. MetAmp is degraded to methionol plus 5'-adenylic acid by 5'-phosphodiesterase. Adenosine deaminase, adenylic acid deaminase and 3':5'-phosphodiesterase cannot use MetAMP as substrate but the last enzyme is inhibited. The presence of MetAMP in cultures provokes a small but reproducible increase in the level of methionyl-tRNA synthetase and 5'-phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Methionyl-tRNA synthetase of Escherichia coli. A zinc metalloprotein.

The native dimeric form of methionyl-tRNA synthetase of Escherichia coli contains two zinc atoms per dimer, one per subunit. The bound zinc is retained upon trypsin modification which yields a monomer with one zinc atom. The enzymatic activity of both the dimeric forms is reversibly inhibited by 1,10-phenanthroline but not by its non-chelating analogues. In addition, the native enzyme binds two Mn2+ per dimer with a binding constant of approx. 70 micron but no binding is observed with the trypsin-modified monomer.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Photocross-linking analysis of the contact surface of tRNA Met in complexes with Escherichia coli methionine:tRNA ligase.

Photoinduced covalent cross-linking has been used to identify a common surface of four methionine-accepting tRNAs which interact specifically with the Escherichia coli methionine:tRNA ligase (EC 6.1.1.10). tRNA--ligase mixtures were irradiated, and the covalently linked complexes were isolated and digested with T1 RNase (Schimmel & Budzik, 1977). The fragments lost from the elution profile of the T1 RNase digest were considered to have been cross-linked to the protein and therefore in intimate contact with the enzyme. Only specific cognate tRNA--ligase pairs produce covalently linked complexes. The four substrate tRNAs used in this study have substantially different sequences, but all showed a common cross-linking pattern, supporting the view that the sites cross-linked to the enzyme reflect the functionally common contact surface rather than particularly photoreactivity regions of tRNA. The cross-linked contact surface is comprised of three regions: (1) the narrow groove of the anticodon stem and its extension into the anticodon loop; (2) the 3' terminal residues; and (3) the 3' side of the "T arm". Unlike previous studies with other tRNAs, the D arm is not involved and significant radiation damage is suffered by the tRNA which must be taken into account in the analysis. The results are consistent with and complement chemical modification studies [Schulman, L. H., & Pelka, H. (1977) Biochemistry 16, 4256].

Amino Acyl-tRNA Synthetases↗
PubMed1976

Methionyl-tRNA synthetase from Escherichia coli: active stoichiometry and stopped-flow analysis of methionyl adenylate formaiton.

Native dimeric methionyl-tRNA synthetase and its monomeric proteolytic fragment are shown to form and to bind 1 mol of methyionyl adenylate per polypeptide chain. Moreover, at 25 degrees C, each monomer of the dimeric native enzyme behaves independently, exhibiting the same parameters for the methionine activation reaction as does the monomeric modified enzyme. These results were obtained using several independent methods including equilibrium and nonequilibrium dialysis, active site and tryptophan fluorescence titrations, and stopped-flow by fluorescence. Stopped-flow resolution of the reversible methionine activation reaction also demonstrates that methionine and ATP-Mg2+ react without coupling to form a ternary enzyme-methionine-ATP-Mg2+ complex. This complex readily converts to enzyme-methionyl approximately adenylate-PP-Mg2+ with a standard free energy close to zero. It is concluded that the uncoupled enzyme-methionine-ATP-Mg2+ complex may resemble the transition state of the reaction at the expense of the additional state of the reaction at the expense of the additional synergistic binding energy provided by reciprocal coupling, within the site, of the methionine molecule with the adenosine and PP-Mg2+ parts of the ATP-Mg2+ molecule (Blanguet, S., Fayat, G., and Waller, J. P. (1975), J. Mol. Biol. 94, 1.).

Adenosine Monophosphate↗
PubMed1979

The stereochemical course of amino acid activation by methionyl- and tyrosyl-tRNA synthetases.

Stereochemical analysis has long been recognised as a powerful tool for elucidating the mechanisms of chemical and enzyme-catalysed reactions. Although much is known about the stereochemical course of reactions at saturated carbon, phosphate and thiophosphate esters whose ligands to phosphorus are also tetrahedrally disposed, are capable in principle of revealing sterochemical information about events at the active site of enzymes that transform such substrates. Nucleotidyl transferases are a group of enzymes which in general selectively use one of the diastereoisomers of a nucleoside 5'(1-thiotriphosphate), such as isomers A and B of adenosine 5'(1-thiotriphosphate), designated ATP alpha S-A and ATP alpha S-B, and allow investigation of the stereochemical course of nucleotidyl transfer. We have developed a simple method based on 31P nuclear magnetic resonance spectroscopy for determining the stereochemical course of these reactions, and using this method show here that the nucleotidyl transfer step in two aminoacyl-tRNA synthetases from Escherichia coli occurs with inversion of configuration at phosphorus. These observations greatly constrain the mechanistic possibilities for these enzymes, and are interpreted most simply as a direct 'in line' transfer from ATP to the amino acid.

Amino Acyl-tRNA Synthetases↗
PubMed1977

The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase.

The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.

Adenosine Triphosphate↗