We have investigated the preequilibrium kinetics of the L-isoleucine activation reaction catalyzed by Ile-tRNA synthetase in the presence of a fluorescent reporter group, 2-p-toluidinylnaphthalene-6-sulfonate, using the stopped-flow technique. It is found that of all the reactants involved, L-isoleucine binds slowest to the enzyme, apparently in a two-step process. The kinetics of the reaction are invariant in the presence of co-reactants, whereas the kinetics for ATP are drastically changed in the presence of Mg{sup 2+} ions. The formation of enzyme bound L-isoleucyl {approx} AMP is conveniently followed at dilute concentrations. The value for the rate constant of formation was determined to be 135 sec{sup -1} and of the reverse process to be 670 sec{sup -1} at pH 8.0 25 C. These values are considerably higher than the rate constant 15 sec{sup -1} of the dissociation reaction for L-isoleucine. The value of the kinetically defined equilibrium constant between the ternary Michaelis-Menten complex and the ternary enzyme-product complex indicates that, at equilibrium, the Michaelis-Menten complex is favored. The effect of temperature has been determined, and a tentative interpretation of the thermodynamic parameters is offered. The zero standard enthalpy and positive entropy for binding of L-isoleucine is consistent with hydrophobic interactions, whereas the enzyme-ligand …

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