Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli

Michel Merle, Veronique Trezeguet, Pierre Vincent Graves, David Andrews, Karl H. Muench, Bernard Labouesse

Research output: Contribution to journalArticle

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Abstract

By gel filtration and titration on DEAE-cellulose filters we show that Escherichia coli tryptophanyl-tRNA synthetase forms tryptophanyl adenylate as an initial reaction product when the enzyme is mixed with ATP-Mg and tryptophan. This reaction precedes the synthesis of the tryptophanyl-ATP ester known to be formed by this enzyme. The stoichiometry of tryptophanyl adenylate synthesis is 2 mol per mole of dimeric enzyme. When this reaction is studied either by the stopped-flow method, by the fluorescence changes of the enzyme, or by radioactive ATP depletion, three successive chemical processes are identified. The first two processes correspond to the synthesis of the two adenylates, at very different rates. The rate constants of tryptophanyl adenylate synthesis are respectively 146 ± 17 s-1 and 3.3 ± 0.9 s-1. The third process is the synthesis of tryptophanyl-ATP, the rate constant of which is 0.025 s-1. The Michaelis constants for ATP and for tryptophan in the activation reaction are respectively 179 ± 35 μM and 23.9 ± 7.9 μM, for the fast site, and 116 ± 45 μM and 3.7 ± 2.2 μM, for the slow site. No synergy between ATP and tryptophan can be evidenced. The data are interpreted as showing positive cooperativity between the subunits associated with conformational changes evidenced by fluorometric methods. The pyrophosphorolysis of tryptophanyl adenylate presents a Michaelian behavior for both sites, and the rate constant of the reverse reaction is 360 ± 10 s-1 with a binding constant of 196 ± 12 μM for inorganic pyrophosphate (PPi). Major differences appear between tryptophanyl-tRNA synthetases from E. coli and from beef. In the procaryotic enzyme, the two subunits present nearly half-of-the-sites reactivity but exhibit kinetic positive cooperativity toward tryptophan. In the eucaryotic enzyme, the two subunits are identically active at substrate saturation but exhibit negative cooperativity toward tryptophan. The bacterial enzyme makes tryptophan derivatives such as tryptophanamide. The mammal enzyme does not. The E. coli enzyme does not cross-react with antibodies raised against the beef protein.

Original languageEnglish
Pages (from-to)1115-1123
Number of pages9
JournalBiochemistry
Volume25
Issue number5
StatePublished - Dec 1 1986
Externally publishedYes

Fingerprint

Tryptophan-tRNA Ligase
Escherichia coli
Tryptophan
Enzymes
Adenosine Triphosphate
Beef
Rate constants
Chemical Phenomena
DEAE-Cellulose
Mammals
Titration
Reaction products
Stoichiometry
Gel Chromatography
Esters
Fluorescence
Gels
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Merle, M., Trezeguet, V., Graves, P. V., Andrews, D., Muench, K. H., & Labouesse, B. (1986). Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli. Biochemistry, 25(5), 1115-1123.

Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli. / Merle, Michel; Trezeguet, Veronique; Graves, Pierre Vincent; Andrews, David; Muench, Karl H.; Labouesse, Bernard.

In: Biochemistry, Vol. 25, No. 5, 01.12.1986, p. 1115-1123.

Research output: Contribution to journalArticle

Merle, M, Trezeguet, V, Graves, PV, Andrews, D, Muench, KH & Labouesse, B 1986, 'Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli', Biochemistry, vol. 25, no. 5, pp. 1115-1123.
Merle M, Trezeguet V, Graves PV, Andrews D, Muench KH, Labouesse B. Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli. Biochemistry. 1986 Dec 1;25(5):1115-1123.
Merle, Michel ; Trezeguet, Veronique ; Graves, Pierre Vincent ; Andrews, David ; Muench, Karl H. ; Labouesse, Bernard. / Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli. In: Biochemistry. 1986 ; Vol. 25, No. 5. pp. 1115-1123.
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abstract = "By gel filtration and titration on DEAE-cellulose filters we show that Escherichia coli tryptophanyl-tRNA synthetase forms tryptophanyl adenylate as an initial reaction product when the enzyme is mixed with ATP-Mg and tryptophan. This reaction precedes the synthesis of the tryptophanyl-ATP ester known to be formed by this enzyme. The stoichiometry of tryptophanyl adenylate synthesis is 2 mol per mole of dimeric enzyme. When this reaction is studied either by the stopped-flow method, by the fluorescence changes of the enzyme, or by radioactive ATP depletion, three successive chemical processes are identified. The first two processes correspond to the synthesis of the two adenylates, at very different rates. The rate constants of tryptophanyl adenylate synthesis are respectively 146 ± 17 s-1 and 3.3 ± 0.9 s-1. The third process is the synthesis of tryptophanyl-ATP, the rate constant of which is 0.025 s-1. The Michaelis constants for ATP and for tryptophan in the activation reaction are respectively 179 ± 35 μM and 23.9 ± 7.9 μM, for the fast site, and 116 ± 45 μM and 3.7 ± 2.2 μM, for the slow site. No synergy between ATP and tryptophan can be evidenced. The data are interpreted as showing positive cooperativity between the subunits associated with conformational changes evidenced by fluorometric methods. The pyrophosphorolysis of tryptophanyl adenylate presents a Michaelian behavior for both sites, and the rate constant of the reverse reaction is 360 ± 10 s-1 with a binding constant of 196 ± 12 μM for inorganic pyrophosphate (PPi). Major differences appear between tryptophanyl-tRNA synthetases from E. coli and from beef. In the procaryotic enzyme, the two subunits present nearly half-of-the-sites reactivity but exhibit kinetic positive cooperativity toward tryptophan. In the eucaryotic enzyme, the two subunits are identically active at substrate saturation but exhibit negative cooperativity toward tryptophan. The bacterial enzyme makes tryptophan derivatives such as tryptophanamide. The mammal enzyme does not. The E. coli enzyme does not cross-react with antibodies raised against the beef protein.",
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N2 - By gel filtration and titration on DEAE-cellulose filters we show that Escherichia coli tryptophanyl-tRNA synthetase forms tryptophanyl adenylate as an initial reaction product when the enzyme is mixed with ATP-Mg and tryptophan. This reaction precedes the synthesis of the tryptophanyl-ATP ester known to be formed by this enzyme. The stoichiometry of tryptophanyl adenylate synthesis is 2 mol per mole of dimeric enzyme. When this reaction is studied either by the stopped-flow method, by the fluorescence changes of the enzyme, or by radioactive ATP depletion, three successive chemical processes are identified. The first two processes correspond to the synthesis of the two adenylates, at very different rates. The rate constants of tryptophanyl adenylate synthesis are respectively 146 ± 17 s-1 and 3.3 ± 0.9 s-1. The third process is the synthesis of tryptophanyl-ATP, the rate constant of which is 0.025 s-1. The Michaelis constants for ATP and for tryptophan in the activation reaction are respectively 179 ± 35 μM and 23.9 ± 7.9 μM, for the fast site, and 116 ± 45 μM and 3.7 ± 2.2 μM, for the slow site. No synergy between ATP and tryptophan can be evidenced. The data are interpreted as showing positive cooperativity between the subunits associated with conformational changes evidenced by fluorometric methods. The pyrophosphorolysis of tryptophanyl adenylate presents a Michaelian behavior for both sites, and the rate constant of the reverse reaction is 360 ± 10 s-1 with a binding constant of 196 ± 12 μM for inorganic pyrophosphate (PPi). Major differences appear between tryptophanyl-tRNA synthetases from E. coli and from beef. In the procaryotic enzyme, the two subunits present nearly half-of-the-sites reactivity but exhibit kinetic positive cooperativity toward tryptophan. In the eucaryotic enzyme, the two subunits are identically active at substrate saturation but exhibit negative cooperativity toward tryptophan. The bacterial enzyme makes tryptophan derivatives such as tryptophanamide. The mammal enzyme does not. The E. coli enzyme does not cross-react with antibodies raised against the beef protein.

AB - By gel filtration and titration on DEAE-cellulose filters we show that Escherichia coli tryptophanyl-tRNA synthetase forms tryptophanyl adenylate as an initial reaction product when the enzyme is mixed with ATP-Mg and tryptophan. This reaction precedes the synthesis of the tryptophanyl-ATP ester known to be formed by this enzyme. The stoichiometry of tryptophanyl adenylate synthesis is 2 mol per mole of dimeric enzyme. When this reaction is studied either by the stopped-flow method, by the fluorescence changes of the enzyme, or by radioactive ATP depletion, three successive chemical processes are identified. The first two processes correspond to the synthesis of the two adenylates, at very different rates. The rate constants of tryptophanyl adenylate synthesis are respectively 146 ± 17 s-1 and 3.3 ± 0.9 s-1. The third process is the synthesis of tryptophanyl-ATP, the rate constant of which is 0.025 s-1. The Michaelis constants for ATP and for tryptophan in the activation reaction are respectively 179 ± 35 μM and 23.9 ± 7.9 μM, for the fast site, and 116 ± 45 μM and 3.7 ± 2.2 μM, for the slow site. No synergy between ATP and tryptophan can be evidenced. The data are interpreted as showing positive cooperativity between the subunits associated with conformational changes evidenced by fluorometric methods. The pyrophosphorolysis of tryptophanyl adenylate presents a Michaelian behavior for both sites, and the rate constant of the reverse reaction is 360 ± 10 s-1 with a binding constant of 196 ± 12 μM for inorganic pyrophosphate (PPi). Major differences appear between tryptophanyl-tRNA synthetases from E. coli and from beef. In the procaryotic enzyme, the two subunits present nearly half-of-the-sites reactivity but exhibit kinetic positive cooperativity toward tryptophan. In the eucaryotic enzyme, the two subunits are identically active at substrate saturation but exhibit negative cooperativity toward tryptophan. The bacterial enzyme makes tryptophan derivatives such as tryptophanamide. The mammal enzyme does not. The E. coli enzyme does not cross-react with antibodies raised against the beef protein.

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