Arginyl-tRNA synthetase from Mycobacterium smegmatis SN2: purification and kinetic mechanism

Abstract

Arginyl-tRNA synthetase [L-Arg: tRNAArg ligase (AMP forming) EC 6.1.1.19] has been purified to homogeneity from Mycobacterium smegmatis SN2. The enzyme is a monomer of molecular weight 56,000. The kinetic patterns obtained by initial velocity and product inhibition studies are consistent with a rapid equilibrium random ter ter mechanism. Polyamines stimulated the formation of arginyl-tRNA, the stimulation being more significant at sub-optimal Mg²⁺ concentrations. Initial velocity studies performed in the presence of sub-optimal Mg²⁺and spermine also indicated that the kinetic mechanism remained sequential random. Various attempts to reveal the formation of enzyme-bound arginyl-adenylate provided no evidence for its existence. The reverse reaction, i.e., the deacylation of arginyl-tRNA, required both AMP and PP₁. This observation is consistent with the mechanism proposed.