The Catalytic Mechanism of Glutamyl‐tRNA Synthetase of Escherichia coli

Abstract

The reaction pathway of tRNAG1" charging by glutamyl-tRNA synthetase of Escherichia coli has been investigated. Comparisons of the absolute rates and of the pH dependence of the aminoacylation of tRNAG1", the [32P]PPi -ATP isotope exchange, and the cleavage of [y3'P]ATP catalyzed by this synthetase show that the overall tRNAG1" charging involves at least two distinct steps, whose rate constants are very different at acidic pH but become similar at alkaline pH. Further comparisons of the rates of the AMP and PPi-dependent deacylation of Glu-tRNAG'" and of ["PIPPi -ATP exchange, and the comparison of the efficiency of tRNAG'" with that of Glu-tRNAG'" plus AMP for the [32P]PPi-ATP exchange show that the reversal of tRNAG'" charging involves at all pH values a slow step, either preceding the fast catalysis of the isotope exchange or succeeding it. Finally the study of the effects of various substrates and end-products (PPi, tRNAG'", AMP and hydroxylamine) on some of these reactions shows that the glutamylation process involves the participation of an intermediate complex in which glutamate is associated via a high-energy bond, and for which PPi and tRNAG'" compete : PPi reacts faster with this intermediate at acidic, and tRNAG1" at alkaline pH. This intermediate can transfer the glutamate to hydroxylamine. These results can be the most easily interpreted in the context of a stepwise pathway of tRNAG1" charging, although some of them are compatible with a concerted pathway involving slow conformational changes.Kinetic studies of consumption of [y3'P]ATP and of [3H]Glu-tRNAG1" synthesis at the early time of the reaction show that, at acidic pH, ATP is cleaved in a stoichiometric amount to the synthetase present without concomitant synthesis of Glu-tRNAG'". The rate of ATP consumption during the first catalytic cycle of the enzyme largely exceeds that at the steady state, where it equals that of tRNAG1" charging during and after the first catalytic cycle. At alkaline pH there is no significant difference in the rates of ATP cleavage and tRNAG1" charging during the first turnover of the enzyme and at the steady state. In addition, no end-product dissociation is rate-determining for the overall tRNAG1" charging neither at acidic nor at alkaline pH. These results indicate that the aminoacylation of tRNAG'" takes place between the cleavage of ATP and the dissociation of the end-products, and that this step is rate-determining for the overall tRNAG1" charging at acidic pH, whereas at alkaline pH both steps, the cleavage of ATP and the synthesis of Glu-tRNAG'", occur at similar rates.Until the late seventies the mechanism of aminoacylation of tRNAs was a subject of large controversy. Two schemes have been proposed. The classic Abbreviations. Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid ; aa, amino acid.Enzymes. Glutamyl-tRNA synthetase (EC 6.1.1.17); inorganic concept of the mechanism involves the initial rapid formation of an enzyme-bound aminoacyl adenylate followed by the...

The Catalytic Mechanism of Glutamyl-tRNA Synthetase of Escherichia coli

“…This role has already been proposed in earlier studies [29,43,44]. We show that, under catalytic conditions, tRNA"'" is required in addition to ATP for the binding of glutamate, ATP and tRNA"'" both being able to bind to the free enzyme.…”

“…The two-step pathway of this reaction has been established previously by other approaches [29,44] and this pathway agrees well with the ordered dissociation of the end-products, PPi being the first [Eqn (I)]. This dissociation step could be a requirement for the catalysis of the transfer step if it were to allow the productive interaction of the tRNA (e.g.…”

“…The Glu-tRNA in the tRNA fraction was then isolated on a short DEAE-cellulose column as described previously [29]. The presence of uncharged tRNAG1" was tested after incubation of a sample of this tRNA fraction in a complete aminoacylation mixture containing [14C]glutamate (120 counts min-' pmol-I).…”

“…We reported similar results for E. coli glutamyl-tRNA synthetase [23]. On the other hand, systematic studies of the pH dependence of various partial reactions, such as the pyrophosphorolysis of exogenous adenylate and the transfer of the amino acid from exogenous adenylate to tRNA catalyzed by E. coli arginyl-tRNA synthetase [24], or the [32P]PPi -ATP exchange, the tRNA charging and the amino-acid-dependent and tRNA-dependent ATP consumption catalyzed by E. coli glutamyl-tRNA [29] and glutaminyltRNA [I71 synthetases, showed that PPi and t RNA compete for a common intermediate: PPi reacts faster with this intermediate at acidic pH, whereas tRNA reacts faster at alkaline pH. For synthetases catalyzing the charging of their tRNA by a ping-pong mechanism this competition occurs on a common binding site [30 -351.…”

Crystal Structure of Glutamyl-Queuosine tRNAAsp Synthetase Complexed with l-Glutamate: Structural Elements Mediating tRNA-Independent Activation of Glutamate and Glutamylation of tRNAAsp Anticodon

“…32 tRNA Glu promotes specific binding of L-Glu to the protein without a change in affinity, but with weaker quenching. 32 The K d value of the GluRS·L-Glu complex in the presence of tRNA agrees with the K m value in tRNA aminoacylation 33,34 (Table 1). In contrast, E. coli GluRS binds L-Glu 10 times weaker in the absence of tRNA Glu than in its presence (K d = 1.0 and 0.1 mM, respectively, and K m = 0.1 and 0.03 mM, respectively, in tRNA aminoacylation and ATP-PPi exchange; Table 1).…”