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

Abstract

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. We measured either the formation of a single labeled aminoacyl-tRNATrp per enzyme subunit or the turnover of labeled aminoacyl-tRNATrp synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute "half-of-the-sites reactivity"); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNATrp under each labeling condition were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate of aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

“…The rate of transfer in GlnRS is thus similar to that found in other tRNA synthetases. Burst kinetics also has been observed previously for both ArgRS and TrpRS enzymes (37,39).…”

“…More recently, intrinsic protein tryptophan fluorescence was used as a probe to examine aminoacyl-tRNA synthesis by TyrRS and TrpRS (26,(40)(41)(42). Among these studies, rate constants for the isolated second step by TrpRS, ArgRS, and TyrRS are in the range of 25-45 sec Ϫ1 , similar to the k chem value of 28 sec Ϫ1 measured here for the two-step reaction by GlnRS (37,39,40,42), whereas the experiments with MetRS and PheRS returned lower values of 2-6 sec Ϫ1 for the transfer step (35)(36). The rate of transfer in GlnRS is thus similar to that found in other tRNA synthetases.…”

“…However, early studies of MetRS, PheRS, TyrRS, ArgRS, and TrpRS used chemical-quench flow to examine aminoacylation with 14 C-labeled amino acid (35)(36)(37)(38)(39). More recently, intrinsic protein tryptophan fluorescence was used as a probe to examine aminoacyl-tRNA synthesis by TyrRS and TrpRS (26,(40)(41)(42).…”

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

“…A detailed pre-steady-state analysis of mutants of tRNA His or HisRS compromised with respect to tRNA identity suggested that, in the complete aminoacylation reaction, formation of aminoacyl adenylate in the second active site is contingent upon a productive aminoacyl transfer reaction in the first (20). These and other data led to the proposal of an alternating site model for HisRS (20) that is analogous to the "flip flop" catalysis suggested for class II PheRS (21,22) and class Ic TrpRS (14). This raises the possibility that the catalytic cycles of dimeric class II enzymes and dimeric class Ic enzymes share some common feature.…”

“…The impact of this potential asymmetry in the activation reaction on the complete TyrRS catalytic cycle remains to be explored. TrpRS also exhibits half-of-sites reactivity, and a detailed analysis of the aminoacyl transfer reaction by presteady state kinetics proposed both random and ordered versions of alternating site catalysis as models of the enzyme (14). In the class II ARSs, the tetrameric SepRS represents the single example where half-of-sites reactivity has been demonstrated experimentally (15).…”