Open AccessThe Mechanism of Reaction of Methionyl‐tRNA Synthetase from Escherichia coli
Author(s) -
Blanquet Sylvain,
Fayat Guy,
Waller JeanPierre,
Iwatsubo Motohiro
Publication year - 1972
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1972.tb19707.x
Subject(s) - adenylate kinase , chemistry , ligand (biochemistry) , methionine , enzyme , titration , fluorescence , quenching (fluorescence) , equilibrium constant , binding site , stereochemistry , biochemistry , inorganic chemistry , amino acid , receptor , quantum mechanics , physics
The interactions of methionyl‐tRNA synthetase, a tetrameric enzyme, with various ligands involved in the methionine‐activation reaction (methionine and analogues, ATP, AMP, PP i , methioninyl adenylate and methionyl adenylate) were examined by equilibrium dialysis, absorbance difference spectroscopy and fluorescence measurements at equilibrium and in a stoppedflow apparatus. Two binding sites for methionine and four for ATP were found by equilibrium dialysis. The number of binding sites and the association constants of these ligands for the enzyme were not affected by the presence of Mg 2+ or EDTA. From ligand‐induced fluorescence and absorbance variations, two binding sites were measured for methioninyl adenylate, a structural analogue of methionyl adenylate. The interaction of methionyl‐tRNA synthetase with most of the ligands investigated gave rise to characteristic fluorescence variations of large amplitude. Thus, saturating concentrations of methioninyl adenylate and methionine enhanced the fluorescence emission by 40% and 26%, respectively, while interaction with chemically synthesized methionyl adenylate resulted in 27% quenching. ATP, AMP or PP i , alone or in combinations, did not affect the emission spectrum. However, when ATP was added to enzyme saturated with methionine, the fluorescence level shifted from the initial value of 126% to 82%. Similary, additon of PP i to enzyme saturated with ATP and methionine raised the level of fluorescence from 82% to 112%. In none of the cases investigated did addition of Mg 2+ or EDTA affect the final level of ligand‐induced fluorescence modifications. Titration experiments were conducted to measure the association constants of the ligands for the enzyme. The rates of formation of methionyl adenylate and of reversal of this reaction by PP i were determined by stopped‐flow measurements. Comparison of reaction rates in the presence of Mg 2+ or EDTA reveals a dramatic enhancement of the rates of both reactions in the presence of Mg 2+ .