MECHANISM OF PHENYLALANYL-TRANSFER RNA-SYNTHETASE OF ESCHERICHIA-COLI-K .10. MODULATION OF CATALYTIC PROPERTIES BY MAGNESIUM

The association of phenylalanyl-tRNA and Mg2+ follows a biphasic concentration dependence as indicated by the active site directed fluorescent indicator 2-p-toluidinyl-naphthalene-6-sulfonate. The macroscopic dissociation constants 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 has been measured by established equilibrium and stopped-flow techniques using 2-p-toluidi-nylnaphthalene-6-sulfonate. At 10 mM Mg2+, the association of enzyme and MgATP is biphasic with dissociation constants 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 a Mg2+-dependent coupling of the synergistic reactions at the two active sites of the 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 Michaelis-Menten constant 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 Michaelis-Menten constant of MgPPj. The effects on the thermodynamics and kinetics parallel the occupancy of the low-affinity Mg2+-binding sites of the enzyme. © 1979, American Chemical Society. All rights reserved.