Mechanism of oligomerization of Escherichia coli carbamoyl phosphate synthetase and modulation by the allosteric effectors. A site-directed mutagenesis study.

We use site-directed mutagenesis to clarify the role of effector-mediated oligomerization changes on the modulation of the activity of Escherichia coli carbamoyl phosphate synthetase (CPS) by its allosteric activator ornithine and its inhibitor UMP. The regulatory domain mutations H975L, L990A and N992A abolished, and N987V decreased CPS oligomerization. The oligomerization domain mutation L421E prevented tetramer but not dimer formation. None of the mutations had drastic effects on enzyme activity or changed the sensitivity or apparent affinity of CPS for ornithine and UMP. Our findings exclude the involvement of oligomerization changes in the control of CPS activity, and show that CPS dimers are formed by the interactions across regulatory domains, and tetramers by the interactions of two dimers across the oligomerization domains. A mechanism for effector-mediated changes of the oligomerization state is proposed.