Mechanism of phenylalanyl-tRNA synthetase of Escherichia coli K 10. Modulation of catalytic properties by magnesium

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Abstract
The association of phenylalanyl-tRNA and Mg2+ follows a biphasic concentration dependence as indicated by the active site directed fluorescent indicator 2-p-toluidinylnaphthalene-6-sulfonate. The macroscopic Kd are 0.16 .+-. 0.03 and 4.1 .+-. 0.5 mM. The effect of Mg2+ on the association of enzyme and MgATP, on the synergistic binding of MgATP and L-phenylalaninol, and on the pre-steady-state synthesis and pyrophosphorolysis of the enzyme-phenylalanyladenylate complex in the absence and the presence of tRNAPhe was measured by established equilibrium and stopped-flow techniques using 2-p-toluidinylnaphthalene-6-sulfonate. At 10 mM Mg2+, the association of enzyme and MgATP is biphasic with Kd of 0.25 .+-. 0.03 and 9.1 .+-. 1.7 mM. At 2 mM Mg2+, a single dissociation constant of 5.0 .+-. 0.5 mM is indicated. The coupling constant of the synergistic reaction is 15 at 1 mM Mg2+ and 290 at 10 mM Mg2+. The Hill constant of the sigmoidal dependence is 3.6. The strengthening of the synergism is believed to reflect on Mg2+-dependent coupling of the synergistic reactions at the two active sites of enzyme, the coupling being negligible at 1 mM and maximal at 10 mM Mg2+. The pre-steady-state rate of adenylate synthesis is accelerated by the presence of Mg2+. The effect is to decrease the value of the Km of MgATP. Another effect is to increase the rate constant when tRNAPhe is present. At subsaturating [MgATP], the [Mg2+] dependence of the observed rate constant is hyperbolical in the absence and sigmoidal (Hill constant, 3.5) in the presence of tRNAPhe. The rate of the pyrophosphorolysis is enhanced by a decrease of the Km of MgPPi. The effects on the thermodynamics and kinetics parallel the occupancy of the low-affinity Mg2+-binding sites of the enzyme.