Isoleucyl‐tRNA Synthetase

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Abstract
Fluorescence properties of purified isoleucyl‐tRNA synthetase isolated from Escherichia coli B have been studied. No changes in the quantum yield, energy or polarisation of the emission were detected in the presence (either individually or in combinations) of the substrates and cofactors required for activation of L‐isoleucine.In 2.5 M urea enzyme activity and intrinsic fluorescence intensity (at 340 nm) each decrease with time, showing similar kinetics and rate constants. The rate of this decay is reduced in the presence of ligands which can bind to the enzyme and the effect has been used to measure dissociation constants for enzyme‐ligand complexes. Values have been obtained for the complexes between enzyme and l‐isoleucine (Kdiss= 25 μM), l‐valine (Kdiss=300μM), ATP(Kdiss= 150 μM) and PPi (Kdiss= 200 μM) at 25°. The effects of ionic strength, and the temperature dependence and urea concentration dependence of l‐isoleucine binding have also been studied. Magnesium ions, which are required for catalysis, do not greatly affect the binding of single substrates, but changes are seen in the presence of ATP and l‐isoleucine together. The magnesium ion concentration dependence of this effect (half‐point about 200 μM) and the equilibrium constant for l‐isoleucine activation (2 μM) have both been measured.The reliability of the methods has been discussed. Results have been interpreted in terms of current theories of amino acid activation. The binding parameters are sufficient to explain the stability of enzyme bound l‐isoleucyladenylate without invoking conformation changes. This is consistent with the absence of substrate induced fluorescence changes. Magnesium effects are explained in terms of reduced electrostatic repulsion between reactants bearing like charges.