Kinetic Anticooperativity in Pre‐Steady‐State Formation of Tryptophanyl Adenylate by Tryptophanyl‐tRNA Synthetase from Beef Pancreas

Abstract

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 LYZ 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. ( 3 978, Eur. J. Biochem. 87, 541 -550) in the steady state of the tRNATrp aminoacylation reaction (10 mM total magnesium in 100 mM KCI 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.

Allostery and conformational free energy changes in human tryptophanyl‐tRNA synthetase from essential dynamics and structure networks

“…It is interesting to note that in Sys3 the community pattern around Trp‐AMP in one of the subunits is more favorable for charging the acceptor stem of tRNA with Trp because of the diminished rigidity around it as compared to the other subunit. This validates the concept of half‐of‐the‐sites‐reactivity for hTrpRS at a molecular level 10, 11…”

“…Human tryptophanyl‐tRNA synthetase (hTrpRS; Supporting Information Figure S1) belongs to class Ic, differing from its bacterial counterparts at various key positions 9. hTrpRS is a functional dimer and has been reported to be functionally asymmetric with half‐of‐the‐sites‐reactivity 10, 11. It has also been shown previously that the long range signaling from the anticodon loop of tRNA to the active site of the protein is very poor along the length of the L‐shaped tRNA 12.…”

Ligand dependent intra and inter subunit communication in human tryptophanyl tRNA synthetase as deduced from the dynamics of structure networks

“…Interestingly, such a correlation of the acceptor stem of tRNA2 is not at all observed, emphasizing the asymmetric behavior of the tRNAs too. These observations are consistent with the earlier results [22][23][24] showing that the dimeric hTrpRS has half-of-the-sites reactivity. It has been suggested that the dimeric hTrpRS can get activation in one subunit and binding of Trp to the other, and the two subunits work in an anti-cooperative manner.…”

“…Furthermore, we find that the results from the shortest path analysis are consistent with the kinetic data, showing that only one monomer of the dimeric hTrpRS operate at a time during aminoacylation. [22][23][24] Results and discussion Structures of hTrpRS complexed with different ligands are available (PDB codes 1R6T, 10 1R6U, 19 2AZX, 11 2AKE 13 and 2DR2 13 ),z but no structure has yet been solved in the presence of both the tRNA Trp and the activated Trp (TrpAMP). We considered the protein structure of hTrpRS bound to tRNA Trp and Trp (PDB code 2DR2) for modeling.…”

Asymmetric Amino Acid Activation by Class II Histidyl-tRNA Synthetase from Escherichia coli

“…When asymmetries have been observed, they have been confined to amino acid binding, or differences in the rate of adenylation reaction measured under pre-steadystate conditions. Among the dimeric class I enzymes, Tyr-and TrpRS bind 1 mol of amino acid in the absence of ATP, and two in its presence (9,37). Similarly, the class II dimeric enzyme PheRS binds only 1 mol of amino acid in the absence of ATP and 2 mol in its presence (38).…”