Phenylalanyl‐tRNA Synthetase from Yeast

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...

Section: Comparison I T Sirlz Olhtv T Rnac'hurging Systemsmentioning

confidence: 99%

The Catalytic Mechanism of Glutamyl‐tRNA Synthetase of Escherichia coli

Kern

Lapointe

1980

“…However, some of the enzymes have shown differences in their mechanisms corresponding to the reaction conditions : e.g. phenylalanyl-tRNA synthetase from yeast shows different orders of substrate addition depending on the tRNA concentration [13], tryptophanyl-tRNA synthetase from beef pancreas depends on the magnesium concentration [I 51.…”

mentioning

confidence: 99%

Isoleucyl‐tRNA Synthetase from Baker's Yeast

Freist

Hahr

Cramer

1981

The order of substrate addition to isoleucyl-tRNA synthetase from baker's yeast has been investigated by steady-state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncompetitively with respect to ATP, namely purineriboside (= nebularin), 3'-deoxy-adenosine (= cordycepin), 8-amino-adenosine and 8-azido-adenosine 5'-triphosphates. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. Additionally, bisubstrate kinetics and product inhibition studies were carried out. The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. The results of the pyrophosphate exchange studies are consistent with formation of E . Ile-AMP . ATP . Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and isoleucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter-ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi-uni uni-bi ping-pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter-ter mechanism.

“…Alternatively these substrates may bind in a random fashion, as reported for valyl-tRNA [32], tyrosyltRNA [72], phenylalanyl-tRNA [73] and isoleucyl-tRNA [35] synthetases from E. coli. Tyrosyl-tRNA synthetase from E. coli [72] and phenylalanyl-tRNA synthetase from yeast [74] do not follow the classical ping-pong mechanism, since they bind the three substrates randomly.…”

Section: Comparison I T Sirlz Olhtv T Rna -C'hurging Systemsmentioning

confidence: 99%

The Catalytic Mechanism of Glutamyl-tRNA Synthetase of Escherichia coli

Kern

Lapointe

2005

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. ...