An investigation of the mechanism of activation of tryptophan by tryptophanyl‐tRNA synthetase from beef pancreas

Abstract

Adenosine (S)‐5′‐[α‐17O,18O2]triphosphate has been synthesized and used to investigate the stereochemical course of activation of tryptophan by tryptophanyl‐tRNA synthetase from beef pancreas. It is shown that the reaction proceeds by displacement of Mg pyrophosphate from MgATP with inversion of configuration at Pot. Tryptophanyl‐tRNA synthetase catalyses positional isotope exchange in adenosine 5′‐[β‐18O2]triphosphate in the presence of tryptophan but not in its absence or in the presence of the competitive inhibitors tryptamine and tryptophanol. These observations eliminate a multi‐adenylyl transfer or dissociative mechanism and in combination with the stereochemical study leave a direct associative ‘in line’ displacement at Pα of MgATP as the only tenable mechanism for the activation of tryptophan by tryptophanyl‐tRNA synthetase.

Crystal structures of Saccharomyces cerevisiae tryptophanyl-tRNA synthetase: new insights into the mechanism of tryptophan activation and implications for anti-fungal drug design

“…As the interactions of Lys195 B of the KMSKS loop and Asn18 B of the TIGN motif (equivalent to the HVGH motif of hTrpRS) with this oxygen atom in the scissile bond are important to compensate the increased negative charge on the atom in the proposed dissociative mechanism for bTrpRS (17), the incapability of Arg162 H to execute a similar function as Lys195 B under an equivalent situation and the replacement of Asn18 B by a weakly charged residue His173 H would substantially reduce the effect and hence our results do not favor a dissociative mechanism. On the other hand, considering that at the transition state P α of ATP may move as close as ∼2 Å to the nucleophilic oxygen (Figure 3B) and simultaneously an interaction forms between Arg162 H and the oxygen bridging P α and P β of ATP (Figure 3D), our data suggest an associative mechanism of Trp activation by eukaryotic TrpRSs, which is in accordance with the conclusion of the previous investigation of the phosphoryl transfer reaction of bovine TrpRS by examination of the primary and secondary oxygen kinetic isotope effects (47). …”

Differential effects of the two amino acid sensing systems, the GCN2 kinase and the mTOR complex 1, on primary human alloreactive CD4+ T-cells

“…In this study, the effects of GCN2 kinase activation or mTORC1 inhibition on primary human alloreactive CD4 + T-cells were compared under the same experimental conditions. The two-way mixed lymphocyte reaction (MLR), as a model of alloreactivity (19), the GCN2 kinase activator, tryptophanol (TRP) (14,20), and the mTORC1 inhibitor, rapamycin (RAP), were used (10). The concentration of TRP was selected to inhibit T-cell proliferation with negligible toxicity according to previous studies (7,13,14).…”

Activation of General Control Nonderepressible-2 Kinase Ameliorates Glucotoxicity in Human Peritoneal Mesothelial Cells, Preserves Their Integrity, and Prevents Mesothelial to Mesenchymal Transition

“…GAPDH activity under normal glucose, normal glucose with tryptophanol, normal glucose with halofuginone, high glucose, high glucose with tryptophanol, and high glucose with halofuginone was 97. 33 Figure 4A). Figure 4B).…”

“…In its turn, the activated kinase phosphorylates the eukaryotic initiation factor-2α (e-IF2α), altering the protein translational program of the cell in the context of the cytoprotective integrated stress response [32]. Considering that, in addition to GCN-2 kinase activation, halofuginone interferes with transforming growth factor-β1 (TGF-β1) signal transduction [31] by inhibiting SMAD3 phosphorylation, we also evaluated the effect of tryptophanol, an experimental substance that activates GCN-2 kinase by inhibiting tryptophanyl-tRNA synthetase [22,33].…”