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. The same behaviour is observed for the pyrophosphorolysis reaction. Both subunits exhibit the same affinity for ATP-Mg in the forward reaction and the same affinity for magnesium pyrophosphate in the backward reaction. On the contrary the formation of tryptophanyl adenylate follows biphasic kinetics when tryptophan concentration is much below saturation. This is independent of ATP-Mg concentration and is the consequence of different affinities of the two subunits for tryptophan as already observed by Graves et al. (1979, Eur. J. Biochem. 96, 509-518) in equilibrium dialysis experiments. A monoadenylate-enzyme complex on one subunit has been prepared. This complex made possible the study of the formation of the second adenylate on the other subunit. The formation of this second adenylate followed first-order kinetics at all ATP-Mg and tryptophan concentrations. The tryptophan concentration dependence of the rate of formation of this second adenylate leads to a Michaelis constant close to the dissociation constant of the low affinity tryptophan site of the enzyme. No isomerization step could be evidenced. The experiments were carried out under two conditions corresponding to those used by Merault et al. (1978. Eur. J. Biochem. 87, 541-550) in the steady state of the tRNATrp aminoacylation reaction (10 mM total magnesium in 100 mM KCl and 1 mM free magnesium ions, both at pH 8.0.25 C). No great difference either in the mechanism or in the dissociation and rate constants was observed but an inhibitory effect of KCl. It is concluded that the enzyme is symmetrical as far as the ATP-Mg and the magnesium pyrophosphate sites are concerned and that the rate of the activation reaction reflects the anticooperative occupancy of the tryptophan sites carried by the two subunits.