Dimeric glutamyl-tRNA synthetases from wheat. Kinetic properties and functional structures.

Abstract

The Michaelis constants in the tRNA aminoacylation reaction have been studied for the three dimeric glutamyl-tRNA synthetases C, P and E. The values were found to be: for tRNA, 0.20 microM, and 0.44 microM; for glutamic acid, 10 microM, 83 microM and 83 microM; for MgATP, 0.46 mM, 0.38 mM and 0.26 mM. MgATP concentrations higher than 2 mM induce pronounced inhibition. The presence of the cognate tRNA is required for [32P]PPi-ATP isotopic exchange. In the absence of tRNA no hyperbolic saturation of the enzymes by glutamic acid occurs in our experimental conditions. Analysis of the enzymic activity as a function of enzyme concentration leads to the conclusion that the active forms are dimers which are in equilibrium with inactive monomers. The values of the dissociation constants Kd were found to be 43 nM, 53 nM and 87 nM for glutamyl-tRNA synthetases C, P and E respectively.