No abstract

Replacement by alanine of Ser-948, Thr-974 and Lys-954 of Escherichia coli carbamoyl phosphate synthetase (CPS) shows that these residues are involved in binding the allosteric inhibitor UMP and the activator IMP. The mutant CPSs are active in vivo and in vitro and exhibit normal activation by ornithine, but the modulation by both UMP and IMP is either lost or diminished. The results demonstrate that the sites for UMP and IMP overlap and that the activator ornithine binds elsewhere. Since the mutated residues were found in the crystal structure of CPS near a bound phosphate, Ser-948, Thr-974 and Lys-954 bind the phosphate moiety of UMP and IMP.z 1999 Federation of European Biochemical Societies.

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. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.