scholarly journals Activation of carbamoyl phosphate synthase by N-acetyl-l-aspartate

1974 ◽  
Vol 143 (1) ◽  
pp. 63-66 ◽  
Author(s):  
Henry Jay Forman ◽  
Ronald Waddell ◽  
Paul B. Hamilton ◽  
Santiago Grisolia
Keyword(s):  
Competitive Inhibition ◽  
Large Excess ◽  
Carbamoyl Phosphate ◽  
High Concentrations ◽  
Phosphate Synthase

Carbamoyl phosphate synthase from liver of both rat and frog, normally dependent on N-acetyl-l-glutamate (on the basis of Km and physiological concentrations) as an activator, was shown to be activated by high concentrations of N-acetyl-l-aspartate. However, the high concentrations of N-acetyl-l-aspartate required for activation produce non-competitive inhibition. Similarly, high concentrations of N-acetyl-l-glutamate, in very large excess of the amount required to activate the enzyme, inhibit. The limit for N-acetyl-l-glutamate as an impurity in N-acetyl-l-aspartate was found to be less than 1 in 5000 parts, far below the 1 in 250 parts needed to produce the activation observed with N-acetyl-l-aspartate.

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.

4217C>A polymorphism in carbamoyl-phosphate synthase 1 gene may not associate with hyperammonemia development during valproic acid-based therapy

2014 ◽  
Vol 108 (6) ◽  
pp. 1046-1051 ◽  
Author(s):  
Kazuyuki Inoue ◽  
Eri Suzuki ◽  
Toshiki Takahashi ◽  
Yoshiaki Yamamoto ◽  
Rei Yazawa ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Carbamoyl Phosphate ◽  
Phosphate Synthase

scholarly journals Structural Basis for Competitive Inhibition of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase fromVibrio cholerae

2015 ◽  
Vol 290 (18) ◽  
pp. 11293-11308 ◽  
Author(s):  
Zeyaul Islam ◽  
Adarsh Kumar ◽  
Suruchi Singh ◽  
Laurent Salmon ◽  
Subramanian Karthikeyan
Keyword(s):  
Competitive Inhibition ◽  
Structural Basis ◽  
Phosphate Synthase

scholarly journals Efficient Cleavage of Conjugates of Drugs or Poisons by Immobilized β-Glucuronidase and Arylsulfatase in Columns

1999 ◽  
Vol 45 (12) ◽  
pp. 2173-2182 ◽  
Author(s):  
Stefan W Toennes ◽  
Hans H Maurer
Keyword(s):  
Enzymatic Hydrolysis ◽  
Storage Stability ◽  
Urine Samples ◽  
Agarose Gel ◽  
Large Excess ◽  
Toxicological Analysis ◽  
Systematic Toxicological Analysis ◽  
Efficient Alternative ◽  
High Concentrations ◽  
Very High

Abstract Background: Cleavage of conjugates is an important step in toxicological analysis, especially of urine samples. The aim of this study was to combine the advantages and to reduce the disadvantages of acid hydrolysis and conventional enzymatic hydrolysis procedures. Methods: β-Glucuronidase (GRD; EC 3.2.1.31) and arylsulfatase (ARS; EC 3.1.6.1) were purified and coimmobilized on an agarose gel matrix and packed into columns. Results: In columns packed with GRD and ARS, the test conjugates 4-nitrophenyl glucuronide and 4-nitrophenyl sulfate added into urine could be completely cleaved within 25 min. Even the relatively stable morphine conjugates could be completely hydrolyzed within 60 min in authentic urine samples. Therefore, an incubation time of 1 h is recommended. Enzyme inhibition by matrix or by rather high concentrations of acetaminophen conjugates was tested and found to be up to 50%. However, a large excess of GRD and ARS was used. The immobilizate columns could be reused for at least 70 incubations and had a storage stability of at least 12 weeks. Carryover of analytes in reused columns could be avoided by rinsing with 200 mL/L methanol in acetate buffer. Thus, five drugs known to be contaminants added in very high concentrations into urine could be completely removed from the columns. A study on the applicability in systematic toxicological analysis showed that 120 different drugs and/or their metabolites could be detected in 35 different authentic urine samples. Conclusions: Use of immobilized and column-packed GRD and ARS is an efficient alternative for the cleavage of urinary conjugates in clinical toxicology.

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.

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