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

Abstract

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.

Understanding carbamoyl phosphate synthetase (CPS1) deficiency by using the recombinantly purified human enzyme: Effects of CPS1 mutations that concentrate in a central domain of unknown function

“…Therefore, the distribution of the mutations does not support the possibility of an increase in the interactions between the UFSDs of different enzyme molecules as the cause for the decreased solubility observed with many of these mutants. This agrees with our observation with E. coli CPS that the UFSD is not involved in dimer formation [54], and with our finding that human CPS1 exists mainly as monomers [10,26].…”

Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site‐directed mutagenesis of ornithine site residues

“…Site‐directed mutagenesis using appropriate mutagenic primers, mutation corroboration by DNA sequencing, and expression and purification of wild type and mutant forms of CPS were carried out as described [6,8]. CPS activity was assayed at 37°C in a solution containing 0.1 M Tris–HCl pH 8.0, 0.1 M KCl, 20 mM KHCO 3 , 10 mM glutamine, 5 mM ATP, 5 mM MgSO 4 , and the indicated concentrations of the effectors, determining after 10 min the amount of CP or ADP produced [8].…”

“…The nature of the allosteric signal or the mechanism of signal transmission to the catalytic machinery has not been clarified. CPS heterodimers associate into oligomers, in part through interactions mediated by the allosteric domain [3], but the possibility that the modulatory signals were transmitted between different CPS molecules was excluded recently using mutations that prevented association [5,6]. Thus, the regulatory signals emerging from the allosteric domain must be transmitted through the interface between this domain and the carbamate phosphorylating domain, which are adjacent in the enzyme structure [3].…”

Interactions of linker proteins with the phycobiliproteins in the phycobilisome substructures of Gloeobacter violaceus

“…The identified proteins were: CpcD1 (gene product of gsr1266) with a coverage of 57% and a score of 82, CpcD2 (product of gene gsr1267) with a coverage of 25% and a score of 115 and ApcC (a gene product of gsr1244) with a coverage of 72% and a score of 241. c shows a 10% acrylamide gel of fractions 7, 9, and 12 of the sucrose gradient shown in a. Bands 1-10 of fraction 7, bands 1-8 of fraction 9, and bands marked with an asterisk in fraction 12 were excised and subjected to LC/ESI/MS/MS analyses (Tables 1, 2 for fractions 7 and 9). The identified proteins in fraction 12 were: RNA polymerase b'subunit (gene product of glr4278) with a coverage of 28% and a score of 1,736, RNA polymerase b subunit (gene product of glr2283) with a coverage of 14% and a score of 764, Carbamoyl-phosphate synthase large subunit (gene product of gll1769, Thoden et al 2004;Mora et al 2002) with a coverage of 3% and a score of 161, ATP synthase gamma (c) subunit (gene product of glr4315) with a coverage of 24% and a score of 256 and serine protease (gene product of glr3741) with a coverage of 12% and a score of 167 Photosynth Res (2010) 106:247-261 251 operon. The evidence for the presence of these small linkers in the G. violaceus PBS came from N-terminal sequence analysis, which was only suggestive due to the fact that in each band there was more than one component (Krogmann et al 2007).…”