scholarly journals Nucleotide ligands protect the inter-domain regions of the multifunctional polypeptide CAD against limited proteolysis, and also stabilize the thermolabile part-reactions of the carbamoyl-phosphate synthase II domains within the CAD polypeptide

1986 ◽  
Vol 236 (2) ◽  
pp. 327-335 ◽  
Author(s):  
E A Carrey
Keyword(s):  
Conformational Stability ◽  
Limited Proteolysis ◽  
Nucleotide Binding ◽  
The Other ◽  
Aspartate Transcarbamoylase ◽  
Biosynthetic Enzymes ◽  
Carbamoyl Phosphate ◽  
Nitrogen Donor ◽  
Phosphate Synthase

Improved methodologies are described which allow the measurement of the part-reactions, with glutamine or ammonia as nitrogen donor, of mammalian carbamoyl-phosphate synthase II (EC 6.3.5.5) through the incorporation of [14C]bicarbonate into either carbamoyl phosphate or carbamoylaspartate. The enzyme is part of the multifunctional polypeptide (CAD) which also comprises the pyrimidine-biosynthetic enzymes aspartate transcarbamoylase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3). The conformational stability of the carbamoyl-phosphate synthase was investigated through the inactivation of the part-reactions which occurred during incubation at 37 degrees C. The domain involved in the removal of the amide N from glutamine was more thermolabile than the ammonia-dependent synthase moiety. The former activity was stabilized in the presence of sodium aspartate or MgATP, whereas the latter was stabilized by MgATP and MgUTP. Binding of MgUTP and MgATP to CAD restricted the initial proteolysis by trypsin and elastase of one or both regions linking the carbamoyl-phosphate synthase domain to the other major domains. A model is described to account for both aspects of nucleotide binding to CAD; these stabilizing effects may be important in the cell, where similar concentrations of nucleotides are found.

scholarly journals Carbamoyl-Phosphate Synthase 1 as a Novel Target of Phomoxanthone A, a Bioactive Fungal Metabolite

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 846
Author(s):  
Sara Ceccacci ◽  
Jana Deitersen ◽  
Matteo Mozzicafreddo ◽  
Elva Morretta ◽  
Peter Proksch ◽  
...  
Keyword(s):  
Limited Proteolysis ◽  
Cellular Respiration ◽  
Carbamoyl Phosphate ◽  
Fungal Metabolite ◽  
Target Interaction ◽  
Interaction Sites ◽  
Transport Chain ◽  
Mitochondrial Toxin ◽  
Novel Target ◽  
Phosphate Synthase

Phomoxanthone A, a bioactive xanthone dimer isolated from the endophytic fungus Phomopsis sp., is a mitochondrial toxin weakening cellular respiration and electron transport chain activity by a fast breakup of the mitochondrial assembly. Here, a multi-disciplinary strategy has been developed and applied for identifying phomoxanthone A target(s) to fully address its mechanism of action, based on drug affinity response target stability and targeted limited proteolysis. Both approaches point to the identification of carbamoyl-phosphate synthase 1 as a major phomoxanthone A target in mitochondria cell lysates, giving also detailed insights into the ligand/target interaction sites by molecular docking and assessing an interesting phomoxanthone A stimulating activity on carbamoyl-phosphate synthase 1. Thus, phomoxanthone A can be regarded as an inspiring molecule for the development of new leads in counteracting hyperammonemia states.

scholarly journals Identification of cysteine residues in carbamoyl-phosphate synthase I with reactivity enhanced by N-acetyl-l-glutamate

1989 ◽  
Vol 260 (2) ◽  
pp. 573-576 ◽  
Author(s):  
K Geschwill ◽  
L Lumper
Keyword(s):  
Limited Proteolysis ◽  
Cysteine Residues ◽  
Carbamoyl Phosphate ◽  
Pig Liver ◽  
Slow Reaction ◽  
Allosteric Activator ◽  
Phosphate Synthase

Carbamoyl-phosphate synthase I (pig liver) is modified at the cysteine residues 1327 and 1337 (numbered according to the rat sequence) in the presence of 5 mM-N-acetyl-L-glutamate with enhanced rate. ATP/Mg2+ (greater than or equal to 5 mM) protects against alkylation of these two cysteines and loss of activity. According to the results obtained by limited proteolysis of monobromobimane-modified carbamoyl-phosphate synthase I, the accessible cysteines 1327 and 1337 are located in the C-terminal 20 kDa domain D of the enzyme. N-Bromoacetyl-L-glutamate is an allosteric activator and inactivates carbamoyl-phosphate synthase in a slow reaction.

scholarly journals Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene

2004 ◽  
Vol 382 (2) ◽  
pp. 463-470 ◽  
Author(s):  
Onard J. L. M. SCHONEVELD ◽  
Ingrid C. GAEMERS ◽  
Atze T. DAS ◽  
Maarten HOOGENKAMP ◽  
Johan RENES ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Distal Enhancer ◽  
Carbamoyl Phosphate ◽  
Gene Encoding ◽  
Response Elements ◽  
Response Unit ◽  
Glucocorticoid Response ◽  
Protein Functions ◽  
Spatial Positioning ◽  
Phosphate Synthase

The GRU (glucocorticoid-response unit) within the distal enhancer of the gene encoding carbamoyl-phosphate synthase, which comprises REs (response elements) for the GR (glucocorticoid receptor) and the liver-enriched transcription factors FoxA (forkhead box A) and C/EBP (CCAAT/enhancer-binding protein), and a binding site for an unknown protein denoted P3, is one of the simplest GRUs described. In this study, we have established that the activity of this GRU depends strongly on the positioning and spacing of its REs. Mutation of the P3 site within the 25 bp FoxA–GR spacer eliminated GRU activity, but the requirement for P3 could be overcome by decreasing the length of this spacer to ≤12 bp, by optimizing the sequence of the REs in the GRU, and by replacing the P3 sequence with a C/EBPβ sequence. With spacers of ≤12 bp, the activity of the GRU depended on the helical orientation of the FoxA and GR REs, with highest activities observed at 2 and 12 bp respectively. Elimination of the 6 bp C/EBP–FoxA spacer also increased GRU activity 2-fold. Together, these results indicate that the spatial positioning of the transcription factors that bind to the GRU determines its activity and that the P3 complex, which binds to the DNA via a 75 kDa protein, functions to facilitate interaction between the FoxA and glucocorticoid response elements when the distance between these transcription factors means that they have difficulties contacting each other.

scholarly journals Effects of lipids on nucleotide inhibition of wheat-germ aspartate transcarbamoylase: evidence of an additional level of control?

1996 ◽  
Vol 313 (2) ◽  
pp. 669-673 ◽  
Author(s):  
Ashan KHAN ◽  
Babur Z. CHOWDHRY ◽  
Robert J. YON
Keyword(s):  
Fatty Acids ◽  
Wheat Germ ◽  
Hill Coefficient ◽  
Aspartate Transcarbamoylase ◽  
Kinetic Effect ◽  
Carbamoyl Phosphate ◽  
Allosteric Mechanism ◽  
Additional Level ◽  
Nucleotide Inhibition ◽  
Metabolic Significance

Wheat-germ aspartate transcarbamoylase, a monofunctional trimer, is strongly inhibited by uridine 5ʹ-monophosphate (UMP), which shows kinetic interactions with the substrate, carbamoyl phosphate, suggesting a classical allosteric mechanism of regulation. Inhibition of the purified enzyme by UMP was amplified in the presence of a variety of ionic lipids at concentrations low enough to preclude denaturation. In the absence of UMP, most of these compounds had no kinetic effect or were slightly activating. Two phospholipids did not show the effect. In a homologous series of fatty acids (C6-C16), the potentiating effect was only seen with homologues greater than C8, reaching a maximum at C12. The effect of dodecanoate (C12) on kinetic cooperativity (UMP as variable ligand) was studied. At each of several fixed concentrations of carbamoyl phosphate, dodecanoate had a pronounced effect on the half-saturating concentration of UMP, which was reduced by about half in every case, indicating substantially tighter binding of UMP. However, dodecanoate had relatively little effect on the kinetic Hill coefficient for the cooperativity of UMP. The possible metabolic significance of these effects is discussed.

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