Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNA(Trp) aminoacylation.

Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.

Binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase from bovine pancreas under pH conditions of maximum activity. Analysis by ultracentrifugation, fluorescence quenching and chemical modification.

The binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase (EC 6.1.1.

Reaction of tryptophanyl-tRNA synthetase from beef pancreas with periodate-oxidized ATP.

Tryptophanyl-tRNA synthetase from beef pancreas reacts with periodate-oxidized ATP according to biphasic kinetics. A rapid phase involves two groups of the protein, presumably lysine side-chains. The slow phase corresponds to the reaction of a larger number of groups.

Kinetic evidence for half-of-the-sites reactivity in tRNATrp aminoacylation by tryptophanyl-tRNA synthetase from beef pancreas.

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-[14C]tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-[14C]tryptophan were used.

Tryptophanyl-tRNA synthetase is a major soluble protein species in bovine pancreas.

Besides their central role in protein synthesis, aminoacyl-tRNA synthetases have been found or thought to be involved in other processes. We present here a study showing that tryptophanyl-tRNA synthetase has a surprising tissular distribution. Indeed, immunochemical determinations showed that in several bovine organs such as liver, kidney and heart, tryptophanyl-tRNA synthetase constitutes, as expected, about 0.

The adenosine triphosphate-pyrophosphate isotopic exchange reaction: a tool for determination of tryptophan.

Quantitative determination of tryptophan at the picomole level is described, using the ATP-[32P]PPi isotopic exchange reaction catalyzed by tryptophanyl-tRNA synthetase. Sensitivity limits of 500 fmol were obtained. The presence of other amino acids at a 1000-fold excess over tryptophan did not interfere significantly with the quantitative determination of tryptophan.

Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli.

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.

Formycin 3' end modified tRNATrp. Recognition by avian myeloblastosis virus reverse transcriptase and primer function.

Primer tRNATrp has been modified at the 3' end by adenosine analogues: 2'deoxyadenosine, 3'deoxyadenosine, 3' amino-3' deoxyadenosine and formycin. Aminoacylation of modified tRNATrp with cognate aminoacyl-tRNA synthetase and primer function for DNA synthesis catalyzed by AMV reverse transcriptase have been studied. The tRNATrp was able to accept tryptophan but did not initiate the DNA synthesis directed by 35S AMV RNA.

Tryptophanamide formation by Escherichia coli tryptophanyl-tRNA synthetase.

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.

Substrate depletion analysis as an approach to the pre-steady-state anticooperative kinetics of aminoacyl adenylate formation by tryptophanyl-tRNA synthetase from beef pancreas.

The formation of tryptophanyl adenylate catalyzed by tryptophanyl-tRNA synthetase from beef pancreas has been studied by stopped-flow analysis under conditions where the concentration of one of the substrates was largely decreasing during the time course of the reaction. Under such conditions a nonlinear regression analysis of the formation of the adenylate (adenylate vs. time curve) at several initial tryptophan and enzyme concentrations gave an accurate determination of both binding constants of this substrate.

Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase.

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl-tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA.

On the mechanism of tRNATrp aminoacylation catalysed by beef tryptophanyl-tRNA synthetase using presteady-state kinetics.

The dimeric tryptophanyl-tRNA synthetase from beef pancreas has been found to activate 2 tryptophans/mol enzyme [Eur. J. Biochem.

Involvement of tRNA in retrovirus expression: biological implications of reverse transcriptase-primer tRNA interactions.

We have previously studied the topographical and functional implications of the recognition of primer tRNATrp by avian retrovirus reverse transcriptase. Here we have presented evidence that the enzyme is able to deacylate beef liver Trp-tRNATrp, provided that 35-S viral RNA is present in the incubation mixture. No effect of dNTPs on this activity was observed.

Kinetic anticooperativity in pre-steady-state formation of tryptophanyl adenylate by tryptophanyl-tRNA synthetase from beef pancreas. A consequence of the tryptophan anticooperative binding.

The kinetics of formation of tryptophanyl adenylate by tryptophanyl-tRNA synthetase from beef pancreas has been followed by stopped-flow, using the quenching of fluorescence of the enzyme linked to the amino acid activation reaction. Both subunits of this alpha 2 enzyme catalyze the adenylate formation. At saturation with substrates the rate constant of the activation reaction is the same for both subunits.

Tryptophanyl-tRNA synthetase from beef pancreas. Spectroscopic analysis of the stoichiometry of formation of the enzyme-tryptophanyl-adenylate complex.

The dimeric enzyme tryptophanyl-tRNA synthetase from beef pancreas catalyses the stoichiometric formation of one mole of tryptophanyl-adenylate per subunit. This formation is associated with optical changes (absorbance, fluorescence, optical rotation) and is confirmed by analytical ultracentrifugation. An equal amplitude of the change is observed for each adenylation site at pH 8.

Anticooperative binding of L-tryptophan to tryptophanyl-tRNA synthetase from beef pancreas. Study at equilibrium by dialysis and changes in spectroscopic properties.

Equilibrium dialysis and gel filtration studies show that tryptophanyl-tRNA synthetase from beef pancreas binds two molecules of L-tryptophan per dimer in an anticooperative way. The binding of tryptophan ellicits a series of spectroscopic changes in the protein as seen by absorbance, fluorescence and circular dichroism. The molar absorption change of the protein-tryptophan system upon formation of the complex is delta epsilon292 = 10 400 +/- 1000 M(-1) cm(-1) per dimer.

The conformational oscillation of delta-chymotrypsin involvement of methionine-192.

In delta-chymotrypsin the reactivity of methionine-192 towards p-nitrophenacyl bromide is strongly reduced when the alpha-amino group of isoleucine-16 has been acetylated. Since acetylation of isoleucine-16 brings delta-chymotrypsin to a conformation similar to its alkaline one this suggests that methionine-192 should present an impaired reactivity in the alkaline conformation of the protein. It is indeed observed that its chemical reactivity as a function of pH depends on the ionization state of the alpha-amino group of isoleucine-16 (pKapp 9 at 15 degrees C) as does the structure of the enzyme.

Structure-activity relationships in tryptophanyl transfer ribonucleic acid synthetase from beef pancreas. Influence of the alkylation of the sulfhydryl groups on the dimer-monomer equilibrium.

Upon reaction with N-ethylmaleimide, tryptophanyl-tRNA synthetase from beef pancreas dissociates into subunits. At pH7, the rate of the dissociation is close to both the reaction rate of the buried--SH groups and the rate of inactivation (Iborra, F., Mourgeon, G.

Molecular aspects of the inactivation of tryptophanyl transfer ribonucleic acid synthetase by N-ethylmaleimide.

The tryptic maps of tryptophanyl-tRNA synthetase from beef pancreas show that the 8 cysteinyl residues of the enzyme subunit are located, 2 by 2, on four different peptides. The kinetics of the incorporation of radioactivity from N-[ethyl-14C]ethylmaleimide into these peptides are compared in this paper with the kinetics of the changes of the catalytic properties of the enzyme occurring during alkylation. This comparison allows the identification of (a) the peptide carrying the cysteinyl residues located on the surface of the molecule, (b) the peptide carrying the deeply buried residues unmasked by the dissociation of the subunits, and (c) the peptide carrying the --SH group located in the vicinity of the binding site of tryptophan.

Multiple forms of tryptophanyl-tRNA synthetase from beef pancreas.

Three different forms of tryptophanyl-tRNA synthetase can be isolated from pancreatic extracts. Structural, immunological and catalytic properties of these various forms have been compared. The native enzyme is a dimeric molecule of molecular weight 108000.