Site-directed mutagenesis of a residue located in the regulatory site of Escherichia coli aspartate transcarbamoylase. Involvement of lysine 94 in effector binding and the allosteric mechanism

Lysine 94 in the regulatory chain of aspartate transcarbamoylase has been changed to a glutamine residue by site-directed mutagenesis. The resulting enzyme is almost insensitive to the activator ATP and shows a substantially reduced response to the feedback inhibitor CTP. Competition experiments indicate that ATP is still able to bind at low concentrations to the regulatory site of the mutant enzyme, even though no stimulation could be detected. When the nucleosides adenosine or cytidine were used, the saturation curves of the mutant and the wild-type enzyme became indistinguishable. Together these results indicate that lysine 94 is strongly involved in the binding of ATP and CTP by interacting specifically with the triphosphate moiety of these nucleotide effectors. Furthermore, unlike the wild-type enzyme, the inhibitory and stimulatory effects in the mutant enzyme are insensitive to changes in aspartate concentrations, implying that the lysine 94 side chain is also involved in the allosteric mechanism of the enzyme.