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The mechanism of action of methionyl-tRNA synthetase from Escherichia coli. 1. Fluorescence studies on tRNAMet binding as a function of ligands, ions and pH.

Titration of native methionyl-tRNA synthetase from Escherichia coli with tRNAfMet and tRNAmMet leads to 37% and 38% quenching of the enzyme fluorescence respectively. The quenching curves for the two tRNAs are not additive. Analysis of the binding data indicates the presence of a single site for tRNA per mole enzyme, with a three-fold higher affinity for tRNAmMet. The tRNA binding parameters do not vary significantly in the presence of a saturating concentration of methionine. On the other hand, titration of the enzyme · methioninyl-adenylate complex (two moles of the ligand bound/mole enzyme) with tRNAfMet and tRNAmMet, leads to an extensive decrease of fluorescence, from an initial level of 134% to 40% and 35%, respectively, indicative of a major structural or conformational change of the enzyme. Analysis of the quenching curves reveals two distinct binding processes, corresponding to occupation of two tRNA sites on the enzyme. 
 
 
 
Requirements for optimal interaction between enzyme and tRNA were investigated. Magnesium ions are essential for complex formation. Maximal interaction is attained at 1 mM and 4 mM MgCl2, respectively, for tRNAmMet and tRNAfMet. However, close to 40 mM MgCl2 are required to reach equivalence of the two tRNA sites which are available in the presence of methioninyl adenylate. Both spermine and spermidine are able to simulate the role of magnesium in the binding of tRNAMet to the free enzyme, but not in the occupation of the second tRNA site on the enzyme · methioninyl-adenylate complex. 
 
 
 
Addition of KCl results in a decrease of the fluorescence quenching produced by tRNA and in a lowering of the affinity for tRNA, both in the presence of MgCl2 and spermidine. Essentially similar quenching levels and affinities are obtained when titration experiments are performed in imidazole-HCl or Tris-HCl buffer in the pH range of 6.3 to 8.7. In contrast, in potassium phosphate buffer which inhibits the enzyme-tRNA interaction, raising the pH leads to a progressive decrease of the fluorescence quenching caused by tRNA, with concomitant lowering of its affinity for the enzyme. 
 
 
 
For comparative purposes the interaction of tRNAMet with trypsin-modified methionyl-tRNA synthetase was investigated. Analysis of the binding data indicates the presence of a single tRNA site per mole enzyme, even in the presence of saturating amounts of methioninyl adenylate (one mole of this ligand bound per mole enzyme). Furthermore, the magnesium ion concentration required to reach maximal quenching is the same (i.e. 4 mM) in the presence or absence of methionyl adenylate.