ISOLEUCYL-tRNA-SYNTHETASE A FLUORESCENCE STUDY OP THE BINDINGPROPERTIES OF THE SYNTHETASE FROM ESCHERICHIA COLI
Fluorescence properties of purified isoleucyl-tRNA-synthetase isolated from E. coli B have been studied. No changes in the quantum yield, energy or polarization 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 (K{sub diss} = 2.5 x 10{sup -5} M), L-valine (K{sub diss} = 3.0 x 10{sup -4} M), ATP (K{sub diss} = 1.5 x 10{sup -4} M) and PP{sub i} (K{sub diss} = 2.0 x 10{sup -4} M) at 25{sup o}. 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 2 x 10{sup -4} M) and the equilibrium constant for L-isoleucine activation (2 x 10{sup -6} M) 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-isoleucylarenylate 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.
- Research Organization:
- Ernest Orlando Lawrence Berkeley NationalLaboratory, Berkeley, CA (US)
- Sponsoring Organization:
- USAEC
- DOE Contract Number:
- AC02-05CH11231
- OSTI ID:
- 925547
- Report Number(s):
- UCRL--20212
- Country of Publication:
- United States
- Language:
- English
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