The Catalytic Mechanism of Glutamyl‐tRNA Synthetase of Escherichia coli

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Abstract
The reaction pathway of tRNAGlu 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 tRNAGlu, the [32P]PPi– ATP isotope exchange, and the cleavage of [γ‐32P]ATP catalyzed by this synthetase show that the overall tRNAGlu 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‐tRNAGlu and of [32P]‐PPi–ATP exchange, and the comparison of the efficiency of tRNAGlu with that of Glu‐tRNAGlu plus AMP for the [32P]PPi– ATP exchange show that the reversal of tRNAGlu 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, tRNAGlu, 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 tRNAGlu compete: PPi reacts faster with this intermediate at acidic, and tRNAGlu 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 tRNAGlu charging, although some of them are compatible with a concerted pathway involving slow conformational changes.Kinetic studies of consumption of [γ‐32P]ATP and of [3H]Glu‐tRNAGlu 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‐tRNAGlu. 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 tRNAGlu charging during and after the first catalytic cycle. At alkaline pH there is no significant difference in the rates of ATP cleavage and tRNAGlu 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 tRNAGlu charging neither at acidic nor at alkaline pH. These results indicate that the aminoacylation of tRNAGlu takes place between the cleavage of ATP and the dissociation of the end‐products, and that this step is rate‐determining for the overall tRNAGlu charging at acidic pH, whereas at alkaline pH both steps, the cleavage of ATP and the synthesis of Glu‐tRNAGlu, occur at similar rates.