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Kinetics of formation of tryptophanyl‐adenylate by tryptophanyl‐tRNA synthetase from beef pancreas
Author(s) -
Merault Guy,
Labouesse Julie,
Graves Pierre-Vincent,
Labouesse Bernard
Publication year - 1981
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(81)80278-5
Subject(s) - chemistry
The formation of an aminoacyl-adenylate has been shown to occur for most aminoacyl-tRNA synthetases as a first step in the tRNA aminoacylation reaction [ 1,2]. In the particular case of tryptophanyl-tRNA synthetase from beef pancreas a tryptophanyl-adenylate-enzyme complex has been evidenced by gel filtration [3] and spectroscopic changes [4]. Two moles of adenylate can be bound per mole of dimeric enzyme, though under some conditions it has been suggested that tryptophan can also be convalently linked to the protein in a 1: 1 ratio when the enzyme is obtained after fast purification procedure [5]. In the case of the non-covalent complex of the enzyme with L-tryptophan 2 mol tryptophan are bound in an apparent anti-cooperative way to the protein [6]. The formation of the adenylate-enzyme complex has not been studied under prestationary conditions which could show whether or not it is rate determining in the overall ATP-PP, isotope exchange and in the tRNA aminoacylation reactions. Since the formation of the adenylate-enzyme complex can be evidenced by spectroscopic changesof the enzyme [4], this reaction can be followed kinetically using the fluorescence changes of the system. This formation can also be studied by the depletion of [j’P]ATP according to [7] and by the measurement of the stoichiometry of appearance of [‘4C]tryptophanyl-adenylate [8] in order to ascertain that the recorded variations of the spectroscopic signal correspond really to the chemical step of carboxyl activation of tryptophan. This paper presents a preliminary report of the study of such a pre-stationary phase.