The catalytic mechanism of glutamyl-tRNA synthetase of Escherichia coli. Evidence for a two-step aminoacylation pathway, and study of the reactivity of the intermediate complex.

Abstract

The sequence of substrate binding and of end-product dissociation at the steady state of the catalytic process of tRNAGlu aminoacylation by glutamyl-tRNA synthetase from Escherichia coli has been investigated using bisubstrate kinetics, dead-end and end-product inhibition studies. The nature of the kinetic patterns indicates that ATP and tRNAGlu bind randomly to the free enzyme, whereas glutamate binds only to the ternary enzyme · tRNAGlu· ATP complex. Binding of ATP to the enzyme hinders that of tRNAGlu and vice versa. After interconversion of the quaternary enzyme · substrates complex the end-products dissociate in the following order: PPi first, AMP second and Glu-tRNA last. In addition to its role as substrate and as effector with ATP for the binding of glutamate, tRNAGlu promotes the catalytically active enzyme state. Whereas at saturating tRNAGlu concentration the catalysis is rate-determining, this conformational change can be rate-determining at low tRNAGlu concentrations. The results are discussed in the light of the two-step aminoacylation pathway catalyzed by this synthetase.