Human and Escherichia coli tryptophanyl-tRNA synthetases have bi-uni-uni-bi ping-pong mechanisms. Both enzymes have ordered binding of substrates, ATP first. These conclusions from steady-state kinetics were supported by data obtained with the inhibitor L-tryptophanamide for both enzymes and with the inhibitor adenosine for the human enzyme. With product inhibition experiments we determined that the E. coli enzyme has ordered product release, AMP first, whereas the human enzyme has random product release. The rate of the L-tryptophan-dependent ATP-[32P]PPi exchange reaction as catalyzed by either enzyme is not altered by the presence of either brewers' yeast tRNA or E. coli tRNA. For both enzymes high concentrations of ATP produced competitive inhibition patterns on Lineweaver-Burk plots derived from experiments with tRNATrp as the variable substrate. The human enzyme synthesizes stoichiometric amounts of tryptophanyl-ATP ester, whereas the enzyme from E. coli synthesizes more than stoichiometric amounts of this unusual product.
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