Phenylalanyl-tRNA synthetase from E.coli: Synergistic coupling between the sites for binding of L-phenylalanine and ATP

Holler, Eggehard; Bartmann, Peter; Hanke, Till; Kosakowski, Heinz M.

doi:10.1016/0006-291x(73)90593-7

Cited by 3 publications

(1 citation statement)

References 14 publications

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“…When the extent of AMP-induced fluorescence quenching (20%) was compared with the total quenching (64%) upon addition of saturating amounts of L-phenylalanine (Figure 4A, at the end of the experiment), it was seen that only a fraction of the enzyme must have been occupied by L-phenylalanine. probably as Lphenylalanyl adenylate (Holler and Calvin, 1972). The apparent discrepancy between this conclusion and the initial liberation of amino acid from tRNA will be dealt with under Discussion.…”

Section: Resultsmentioning

confidence: 90%

Equilibrium analysis of L-Phe-tRNA^Phe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10

Bartmann¹,

Hanke²,

Hammer-Raber³

et al. 1974

Biochemistry

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Section: Resultsmentioning

confidence: 90%

Equilibrium analysis of L-Phe-tRNA^Phe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10

Bartmann¹,

Hanke²,

Hammer-Raber³

et al. 1974

Biochemistry

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Valyl-tRNA synthetase from baker's yeast. Ligand binding properties and stability of the enzyme-bound adenylate

Kern¹,

Giegé²,

Ebel³

1981

Biochemistry

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The Mechanism of Action of Methionyl-tRNA Synthetase from Escherichia coli. Equilibrium-Dialysis Studies on the Binding of Methionine, ATP and ATP-Mg2+ by the Native and Trypsin-Modified Enzymes

Fayat

Waller

1974

Eur J Biochem

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A comparative study of the interactions of native and trypsin-modified methionyl-tRNA synthetases with methionine and ATP was undertaken.I n imidazole buffer in the absence of MgCl,, the trypsin-modified enzyme possesses one binding site for methionine and one for ATP ( K A = 20 mM-l and 5.5 mM-1, respectively). Binding of each substrate is abolished in the presence of methioninyl adenylate, thereby implicating these sites in adenylate formation. Binding of ATP, but not of methionine, is strongly inhibited by phosphate ions. Furthermore, the unique ATP site on the trypsin-modified enzyme strongly discriminates against ATP-Mg2+ ( K A < 0.7 mM-l). However, in the presence of structural analogues of methionine (methioninol and 3-methylthiopropylamine), the enzyme acquires the ability to interact efficiently with ATP-Mga+ ( K A = 10 mM-l).Native methionyl-tRNA synthetase possesses two sites for methionine and four for ATP. Occupation of the methionine sites and of two out of the four ATP sites by their respective ligands, is abolished in the presence of methioninyl adenylate, indicating that these sites are involved in adenylate formation.The two non-equivalent catalytic ATP sites (A sites) are sbrongly inhibited by phosphate ions and display a lower affinity for ATP-Mg2+ than for ATP. The ability of these sites to interact with ATP-Mg2+ is restored in the presence of analogues of methionine, as in the case of the trypsinmodified enzyme. Conversely, the two equivalent non-catalytic ATP sites (B sites) are unaffected by the presence of phosphate or magnesium ions.The implications of these findings on the mechanism of action of methionyl-tRNA synthetase are discussed.Native methionyl-tRNA synthetase from EscheTichia coli has a molecular weight of about 175000 [l -41. Earlier studies, based on dissociation by 8 M urea [2,5] or maleic anhydride [3], both at pH 9, had led to the conclusion that the enzyme is composed of four, probably identical subunits of molecular weight about 45000. However, recent studies by Koch (personal communication), involving quantitative carboxyl-terminal determinations, show that the subunit has a molecular weight of about 90000, a finding corroborated by acrylamide-gel electrophoresis in dodecylsulfate [7] (and Koch, G., personal communication). While these results establish the dimeric structure of the native enzyme, aminoacid sequence studies and fingerprinting performed Enzyme. L-Methionyl-tRNA synthetase or L-methionine tRNAMet ligase (AMP-forming) (EC 6.1.1.10).by Bruton [S] only reveal peptides accounting for a molecular weight of about 45000. These findings indicate that the subunit of 90000 molecular weight may contain a high degree of duplication [S] and suggest that the four similar "subunits" observed earlier, after treatment with 8 M urea or maleic anhydride at alkaline pH, may have resulted from chain cleavage of each original subunit into two similar fragments. The latter suggestion is supported by the finding that in 8 M urea at pH 7.5, the enzyme does not dissociate beyond t...

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Phenylalanyl-tRNA synthetase from E.coli: Synergistic coupling between the sites for binding of L-phenylalanine and ATP

Cited by 3 publications

References 14 publications

Equilibrium analysis of L-Phe-tRNA^Phe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10

Equilibrium analysis of L-Phe-tRNA^Phe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10

Valyl-tRNA synthetase from baker's yeast. Ligand binding properties and stability of the enzyme-bound adenylate

The Mechanism of Action of Methionyl-tRNA Synthetase from Escherichia coli. Equilibrium-Dialysis Studies on the Binding of Methionine, ATP and ATP-Mg2+ by the Native and Trypsin-Modified Enzymes

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Phenylalanyl-tRNA synthetase from E.coli: Synergistic coupling between the sites for binding of L-phenylalanine and ATP

Cited by 3 publications

References 14 publications

Equilibrium analysis of L-Phe-tRNAPhe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10

Equilibrium analysis of L-Phe-tRNAPhe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10

Valyl-tRNA synthetase from baker's yeast. Ligand binding properties and stability of the enzyme-bound adenylate

The Mechanism of Action of Methionyl-tRNA Synthetase from Escherichia coli. Equilibrium-Dialysis Studies on the Binding of Methionine, ATP and ATP-Mg2+ by the Native and Trypsin-Modified Enzymes

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Equilibrium analysis of L-Phe-tRNA^Phe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10

Equilibrium analysis of L-Phe-tRNA^Phe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10