The Mechanism of Action of Methionyl‐tRNA Synthetase from Escherichia coli

The complete rate equation for the ATP‐PP 1 exchange reaction at equilibrium catalyzed by native and trypsin‐modified methionyl‐tRNA synthetases was determined. The method does not differentiate between the two forms of the enzyme. In both cases, the detection of enzyme · methionine and enzyme · ATP‐Mg 2+ complexes demonstrates random order of binding of the substrates. Furthermore the parameters of the rate equation have been calculated and are found to be very similar. It is shown that the equilibrium constant for ATP‐Mg 2+ is of the order of 1.2 mM for both enzymes, in agreement with the predictions from equilibrium dialysis. Under the same standard conditions, spectrofluorimetric analysis at equilibrium of the reactions of methionine with the enzyme or with the enzyme · ATP‐Mg 2+ complex, of ATP‐Mg 2+ with the enzyme · methionine complex and of PP 1 ‐Mg 2+ with the enzyme · methionyl adenylate complex was performed. The results obtained provide additional evidence for the similarity of the two forms of the enzyme and confirm the rate equation for the ATP‐PP 1 exchange reaction. Both approaches indicate a stoichiometry of two and one catalytic sites per mole enzyme for native and trypsin‐modified methionyl‐tRNA synthetases, respectively.