The mechanism of action of methionyl-tRNA synthetase from Escherichia coli. Equilibrium-dialysis studies on the binding of methionine, ATP and ATP-Mg2+ by the native and trypsin-modified enzymes.

Abstract

A comparative study of the interactions of native and trypsin-modified methionyl-tRNA synthetases with methionine and ATP was undertaken. In imidazole buffer in the absence of MgCl2, the trypsin-modified enzyme possesses one binding site for methionine and one for ATP (KA= 20 mM−1 and 5.5 mM−1, respectively). Binding of each substrate is abolished in the presence of methioninyl adenylate, thereby implicating these sites in adenylate formation. Binding of ATP, but not of methionine, is strongly inhibited by phosphate ions. Furthermore, the unique ATP site on the trypsin-modified enzyme strongly discriminates against ATP-Mg2+ (KA≤ 0.7 mM−1). However, in the presence of structural analogues of methionine (methioninol and 3-methylthiopropylamine), the enzyme acquires the ability to interact efficiently with ATP-Mg2+ (KA= 10 mM−1). Native methionyl-tRNA synthetase possesses two sites for methionine and four for ATP. Occupation of the methionine sites and of two out of the four ATP sites by their respective ligands, is abolished in the presence of methioninyl adenylate, indicating that these sites are involved in adenylate formation. The two non-equivalent catalytic ATP sites (A sites) are strongly inhibited by phosphate ions and display a lower affinity for ATP-Mg2+ than for ATP. The ability of these sites to interact with ATP-Mg2+ is restored in the presence of analogues of methionine, as in the case of the trypsin-modified enzyme. Conversely, the two equivalent non-catalytic ATP sites (B sites) are unaffected by the presence of phosphate or magnesium ions. The implications of these findings on the mechanism of action of methionyl-tRNA synthetase are discussed.