ISOLEUCYL-tRNA SYNTHETASE OF E. coli B. A RAPID KINETIC INVESTIGATION OF THE L-ISOLEUCINE ACTIVATING REACTION

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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 ... continued below

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52 p.

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Holler, E. & Calvin, Melvin May 1, 1972.

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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 complexes for ATP and pyrophosphate might be stabilized by hydrogen-bonds and ion-ion interactions. The equilibrium constant of formation of the ternary enzyme-product complex from the Michaelis-Menten complex does not increase significantly with temperature. The types of kinetic pathways have been restricted to the alternative of a random mechanism or an ordered sequential mechanism in which L-iso-leucine binds first. We believe that the mechanism is random.

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52 p.

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  • Journal Name: Biochemistry

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  • Report No.: LBL-939
  • Grant Number: DE-AC02-05CH11231
  • DOI: 10.1021/bi00770a012 | External Link
  • Office of Scientific & Technical Information Report Number: 942297
  • Archival Resource Key: ark:/67531/metadc894313

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  • May 1, 1972

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  • Sept. 27, 2016, 1:39 a.m.

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  • Sept. 30, 2016, 6:37 p.m.

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Holler, E. & Calvin, Melvin. ISOLEUCYL-tRNA SYNTHETASE OF E. coli B. A RAPID KINETIC INVESTIGATION OF THE L-ISOLEUCINE ACTIVATING REACTION, article, May 1, 1972; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc894313/: accessed September 22, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.