The dimeric tryptophanyl‐tRNA synthetase from beef pancreas has been found to activate 2 tryptophans/mol enzyme Eur. J. Biochem. (1982) 128, 389–398. By using quenched‐flow and stopped‐flow methods ...under presteady‐state conditions, we show that only one enzyme subunit operates at a time in the aminoacylation of tRNATrp and that the transfer reaction is not the rate‐limiting step in the overall aminoacylation process.
When tryptophanyl‐tRNA synthetase from Escherichia coli is allowed to react with L‐tryptophan and ATP‐Mg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture ...reveals three or four sequential processes, depending on the buffer used. Quenched‐flow and stopped‐flow experiments show that the first two processes, which occur in the 0.001–1.0‐s time scale, can be correlated to the formation of two moles of tryptophanyl‐adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl‐ATP ester, as demonstrated by thin‐layer chromatography. This compound has been previously shown to be formed by tryptophanyl‐tRNA synthetase from E. coli K. H. Muench (1969) Biochemistry 8, 4872–4879. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non‐enzymatic hydrolysis of tryptophanyl‐ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl‐adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by high‐performance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl‐ATP ester and is enzyme‐catalyzed with a Km of 1 mM for ammonia and a rate constant of 5.7 min−1 at pH 8.3, 25°C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme‐product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.
The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flow method. The transfer of ...tryptophan to tRNATrp was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-14Ctryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-14Ctryptophan were used. We measured either the formation of a single labeled aminoacyl-tRNATrp per enzyme subunit or the turnover of labeled aminoacyl-tRNATrp synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute "half-of-the-sites reactivity"); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNATrp under each labeling condition were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate of aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.
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 microM and 23.9 +/- 7.9 microM, for the fast site, and 116 +/- 45 microM and 3.7 +/- 2.2 microM, 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 microM for inorganic pyrophosphate (PPi).
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