Antico-operative binding of bacterial and mammalian initiator tRNA Met to methionyl-tRNA synthetase from Escherichia coli

Abstract

The reaction scheme of methionyl-tRNA synthetase from Escherichia coli with the initiator tRNA s Met from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, k R, and for the dissociation, k D, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical k R values of 250 μ m −1 s −1 reflect similar behaviour for antico-operative binding of both tRNA s Met to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNA s Met to measure the mode of exchange of these tRNAs antico-operatively bound to the enzyme. Analysis of the results reveals that antico-operativity does not arise from structural asymmetric assembly of the enzyme subunits. Indeed, both subunits can potentially bind a tRNA molecule. Exchange between tRNA molecules can occur via a transient complex in which both sites are occupied. Either strong and weak sites reciprocate between subunits on the transient complex or occupation of the weak site induces symmetry of this complex. While in the present case, these two alternatives are kinetically indistinguishable, they do account for the observation that, upon increasing the concentration of the competing mammalian tRNA, the rate of exchange of the E. coli initiator tRNA Met is enhanced, due to its faster rate of dissociation from the transient complex. Finally, it has been verified that in the case of the trypsin-modified methionyl-tRNA synthetase which cannot provide more than one binding site for tRNA, exchange of enzymebound bacterial tRNA by mammalian tRNA does proceed to a limiting rate independent of the mammalian tRNA concentration present in the solution.