The sequence of substrate binding and of end-product dissociation at the steady state of the catalytic process of tRNAG'" aminoacylation by glutamyl-tRNA synthetase from Escherichiu coli has been investigated using bisubstrate kinetics, dead-end and end-product inhibition studies. The nature of the kinetic patterns indicates that ATP and tRNAG'" bind randomly to the free enzyme, whereas glutamate binds only to the ternary enzyme . tRNAGL" . ATP complex. Binding of ATP to the enzyme hinders that of tRNA"" and vice versa. After interconversion of the quaternary enzyme . substrates complex the end-products dissociate in the following order: PPi first, AMP second and Glu-tRNA last.In addition to its role as substrate and as effector with ATP for the binding of glutamate, tRNA"'" promotes the catalytically active enzyme state. Whereas at saturating tRNAG'" concentration the catalysis is rate-determining, this conformational change can be rate-determining at low tRNAG'" concentrations.The results are discussed in the light of the two-step aminoacylation pathway catalyzed by this synthetase.The aminoacyl-tRNA synthetases are a group of enzymes which catalyze the specific attachment of amino acids to their cognate tRNAs. This step is crucial for an accurate translation of the genetic code. For this reason the mechanism used by the synthetases to ensure faithful esterification of amino acids to the proper tRNAs has been the focus of many years of intensive study (see [l -51 and references therein).These enzymes catalyze the esterification of the tRNAs via a reaction in which three substrates (ATP, amino acid and tRNA3 are converted into three end-products (AMP, PP, and aminoacyl-tRNA). However, until the late seventies it was not possible to define a general mechanism of tRNA charging for all synthetases (see the general reviews [6-91. The major difficulty resulted from the fact that there are two classes of synthetases, which are characterized by strong differences in their catalytic properties. The first class comprises 17 of the 20 synthetases. These synthetases catalyze a [32P]PPI-ATP exchange in the absence of tRNA. Since, for all enzymes of this group studied up to date, an enzymeadenylate intermediate able to transfer the activated amino acid to the tRNA can be isolated, a two-step aminoacylation pathway, involving the enzyme-adenylate as an obligatory intermediate, has first been proposed [6 -91 ; Eqn (1) :Arginyl [lo-131, glutamyl [14,15], and glutaniinyl [16, 171 tRNA synthetases of various organisms constitute a second group of synthetases characterized by their inability Enzymes. Glutamyl-tRNA synthetase (EC 6.1.1.17).to catalyze the [32P]PPi-ATP exchange in the absence of tRNA [lo-171. As no enzyme-adenylate complexes could be isolated for these enzymes by the usual approaches, a concerted pathway for tRNA charging was proposed, which would apply to these synthetases at least [18,19]; Eqn (2):These two alternative pathways for tRNA charging focused much attention on the detailed mechanisms of these enzymes. ...