Catalytic mechanism of amino acid:tRNA ligases. Synergism and formation of the ternary enzyme-amino acid-ATP complex

Abstract

Formation of binary and ternary enzyme-ligand complexes was investigated for amino acid:tRNA ligases specific for L-isoleucine, L-leucine, and L-phenylalanine. Each of the enzymes exhibited synergistic binding when a substrate was substituted by a structurally related compound. The strength of coupling between the sites binding the amino acid and ATP was strongly dependent on the structure of ligands. The phenomenon was observed with the L-leucine and L-phenylalanine-specific enzymes only in the presence of magnesium. Spermine was inhibitory for L-phenylalanine:tRNA ligase. From the variation which structure of the strength of the observed synergism a correlation scheme was derived considering the ammonium group, the carboxylate group and the side chain of the amino acid, and the adenosine and triphosphate moieties of ATP. The strength of coupling between the subsites binding various combinations of these moieties was evaluated. We found that binding of the subgroups of the amino acid exerts an intramolecular synergism. The strength intramolecular synergism was similar to the strength of the intermolecular synergism observed for the simultaneous binding of an amino alcohol and ATP (or MgATP-2-). We have derived a molecular mechanism for the formation of the ternary enzyme-amino acid-ATP (or MgATP-2-) complex taking into account the synergistic phenomena. The complex is considered to involve electrostatic repulsion between the amino acid carboxylate and the ATP triphosphate moieties. When one of the negatively charged groups have been eliminated, the enzymatic rearrangement which facilitates the formation of this complex may be seen as a synergistic coupling.

Crystal structure of aspartyl-tRNA synthetase from Pyrococcus kodakaraensis KOD: archaeon specificity and catalytic mechanism of adenylate formation

“…This feature should result in a positive coupling which might at least partly compensate for the electrostatic repulsion between the negative charges of the attacking carboxylate group and the receiving α‐phosphoryl, thereby preparing the catalytic step. Such positive couplings were shown biochemically in several class I and class II aminoacyl‐tRNA synthetases (Blanquet et al ., 1975; Holler et al ., 1975). The involvement of these residues in the stabilization of the transition state of the reaction is supported further by the analysis of the two possible conformations of Ser364 observed in the pkAspRS–ATP‐Mg 2+ complex.…”

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

“…The available date on oligomeric synthetases obtained with the tyrosine enzyme from E. coli [9], the methionine enzyme from E. coli [S] and B. steurothermophilus [9,10], the phenylalanine enzyme from E. coli [39] have lead to the conclusions that half-of-the-sites reactivity or michaelian behaviour are exhibited. In some cases an isomerisation step was suggested from the existence of a two-step process [lo, 111.…”

“…Up to now very few studies on aminoacyl-tRNA synthetases have been carried out by pre-steady-state kinetics. The available date on oligomeric synthetases obtained with the tyrosine enzyme from E. coli [9], the methionine enzyme from E. coli [S] and B. steurothermophilus [9,10], the phenylalanine enzyme from E. coli [39] have lead to the conclusions that half-of-the-sites reactivity or michaelian behaviour are exhibited. In some cases an isomerisation step was suggested from the existence of a two-step process [lo, 111. No kinetic cooperativity either positive or negative has been so far observed in relation with the substrate binding properties of the dimeric synthetases when both catalytic sites have been shown to be effectively active.…”

Modification of l-Isoleucyl-tRNA Synthetase with l-Isoleucyl-Bromomethyl Ketone. The Effect on the Catalytic Steps

“…L-Isoleucyl-tRNA synthetase is known to exhibit synergistic binding with certain ligands like L-isoleucinol and ATP [27]. As is seen from Table 2, the stability of the binary synthetase .…”

“…They propose that a linkage between the L-isoleucine catalytic site and the tRNA binding site has been disrupted. (c) Rearrangement subsequent to ligand binding has been suggested from kinetic [23], equilibrium binding [27] and calorimetric work [32]. Further work is being pursued to exclude simple steric hindrance.…”