Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation

Abstract

The adenylation of phenylalanine catalyzed by phenylalanyl-tRNA synthetase was investigated in the absence of tRNA, by rapid kinetic measurements using 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS) as a nonspecific fluorescent reporter group. It is shown that each protomer of the enzyme is able to catalyze independently the adenylation of phenylalanine by ATP, as well as the reversion by pyrophosphate, at least in the absence of tRNA. The kinetic rate constants of synthesis and pyrophosphorolysis are respectively found equal to 100 +/- 20 s-1 and 150 +/- 50 s-1. The symmetric behavior of the enzyme is consistent with a symmetric binding of 2 mol of phenylalanine to the enzyme as shown by equilibrium dialysis experiments. The affinity of phenylalanyladenylate for the enzyme could be characterized by an equilibrium constant of 0.2 x 10(9) M-1.

A peculiar property of aspartyl-tRNA synthetase from bakers' yeast: chemical modification of the protein by the enzymically synthesized aminoacyl adenylate

“…For most of them, an enzymeaminoacyl adenylate complex can easily be isolated by gel filtration of the amino acid activation system containing the synthetase, the cognate amino acid, ATP, and MgCl2 [e.g., ]. Generally, because of the high affinity of aminoacyl adenylates for the synthetases [e.g., Baltzinger et al (1983)] and when inorganic pyrophosphatase is present (to displace the equilibrium completely toward the end products), the complex can be isolated in a 1:1 (adenylate to site) stoichiometry [e.g., ]. When such an experiment is performed with aspartyl-tRNA synthetase [possessing two tRNA sites (Giege et al, 1982) and two amino acid activation sites (unpublished results)], the filtration of the aspartic acid activation system through a Sephadex G-100 column results in a poor recovery of labeled amino acid in the enzyme fraction as compared to the amount expected assuming that both sites are saturated by adenylate.…”

“…in the absence of tRNA, and strong arguments for its participation in the overall tRNA charging process have been reported Fersht & Kaethner, 1978;Fasiolp & Fersht, 1978; Mulvey & Fersht, 1978; Kern & Gangloff, 1981). However, for several systems, starting from preformed enzyme-aminoacyl adenylate complex, incomplete transfers of the activated amino acid to the tRNA have been reported despite the high affinity of the intermediate for the synthetase Baltzinger et al, 1983). It has been shown that part of the enzyme-bound adenylate hydrolyzes after association of the cognate tRNA (Fersht & Jakes, 1975; ).…”

Fast kinetic study of yeast phenylalanyl-tRNA synthetase: an efficient discrimination between tyrosine and phenylalanine at the level of the aminoacyladenylate-enzyme complex

“…The affinity of the noncognate tyrosyladenylate for phenylalanyl-tRNA synthetase and the kinetic binding and dissociation constants have been measured with chemically synthesized tyrosyladenylate. The values obtained are compared to those measured in the cognate system (Baltzinger et al, 1983). The discrimination mechanism is discussed.…”

tRNAGlu Increases the Affinity of Glutamyl-tRNA Synthetase for Its Inhibitor Glutamyl-Sulfamoyl-Adenosine, an Analogue of the Aminoacylation Reaction Intermediate Glutamyl-AMP: Mechanistic and Evolutionary Implications

“…The 50-fold decrease in the affinity of GluRS for Glu-AMS in the absence of tRNA Glu ( Table 1 and Fig 2 ) suggests that the Glu-AMP GluRS interaction in the absence of tRNA Glu is much weaker than that between other aaRSs and their cognate aa-AMP, and has the same order of magnitude as the interaction between a non-cognate aa-AMP and an aaRS, such as tyrosyl-AMP (Tyr-AMP) and phenylalanyl-tRNA synthetase (PheRS) ( Table 3 ). [ 7 , 45 – 48 ] The released intermediate would likely be hydrolyzed very fast by one of the mechanisms of pre-transfer editing [ 49 ] (reviewed by Ling et al, 2009) [ 50 ]. This putative low affinity of GluRS for Glu-AMP could explain why this enzyme evolved to require the presence of its cognate tRNA to activate glutamate, allowing the immediate transfer of glutamate from Glu-AMP to the acceptor end of tRNA, and thus preventing unproductive cleavage of ATP.…”