Kinetics of anticooperative binding of phenylalanyl-tRNAPhe and tRNAPhe to phenylalanyl-tRNA synthetase of Escherichia coli K10.

The kinetics of binding of Phe-tRNAPhe to phenylalanyl-tRNA synthetase were investigated by stopped-flow techniques employing 2-(p-toluidinyl)naphthalene-6-sulfonate as a reporter group. The kinetics were found to follow the concentration of Phe-tRNAPhe in terms of a saturation function. When added, tRNAPhe inhibited both the kinetics of association and the kinetics of dissociation. The kinetics of Phe-tRNAPhe binding have been analyzed to be a superposition of two reactions, the association to the phenylalanine-specific binding site and the association to the tRNA-specific binding site of the enzyme. The two modes are mutually exclusive. When tRNAPhe is added, the binding of Phe-tRNAPhe at the tRNA-specific site is suppressed by competitive inhibition. In binding to the tRNA-specific site, the unacylated tRNA affects the rates of association and dissociation of Phe-tRNAPhe binding at the Phe-specific site in terms of an antagonistic mechanism. The values of both rate constants are decreased depending on the degree of saturation of the enzyme with the unacylated tRNA before and after the association of Phe-tRNAPhe. A model is presented, and kinetic and equilibrium constants are determined.