Kinetics and Reaction Mechanism of the Carbamylphosphate Synthetase of a Multienzyme Aggregate from Yeast

1975 ◽  
Vol 53 (6) ◽  
pp. 721-730 ◽  
Author(s):  
David M. Aitken ◽  
Peter F. Lue ◽  
J. Gordin Kaplan
Keyword(s):  
Reaction Mechanism ◽  
Feedback Inhibition ◽  
Enzyme Reaction ◽  
Competitive Inhibitor ◽  
Potassium Ions ◽  
Ammonium Ions ◽  
Nitrogen Donor ◽  
Sequential Addition ◽  
A Site ◽  
Carbamylphosphate Synthetase

We have studied the kinetics and reaction mechanism of the carbamylphosphate synthetase of an enzyme aggregate functioning in the pyrimidine pathway of yeast. Mg–ATP was found to be one of the substrates of the enzyme reaction which was activated by free Mg2+ and inhibited by free ATP. Feedback inhibition by UTP was non-competitive with respect to both glutamine and bicarbonate. Potassium ions were essential for activity and could not be replaced by sodium. Glutamine could be replaced partially by ammonium ions as nitrogen donor. A bicarbonate-dependent cleavage of ATP was shown to take place in the absence of L-glutamine; L-glutamate was a competitive inhibitor of L-glutamine and the enzyme was shown to synthesize ATP when incubated with ADP and carbamyl phosphate. The reaction mechanism was found to involve sequential addition of the substrates bicarbonate and Mg–ATP and release of ADP, followed by addition of the third substrate glutamine. The purine nucleotide XMP had a pronounced activating effect on the enzyme, acting at a site different from that of UTP. Saturating levels of Mg–ATP eliminated this activation.

Ground water quality of selected areas of Punjab and Sind Provinces, Pakistan: Chemical and microbiological aspects

2019 ◽  
Author(s):  
Chem Int
Keyword(s):  
Ionic Concentration ◽  
Chloride Ions ◽  
Potassium Ions ◽  
Ammonium Ions ◽  
High Concentration ◽  
Phosphate Ions ◽  
Sodium Magnesium ◽  
Very High ◽  
Microbiological Aspects

The assessment of groundwater is essential for the estimation of suitability of water for safe use. An attempt has been made to study the groundwater of selected areas of Punjab (Sheikhupura & Sahiwal) and Sindh (Sindh, Jawar Dharki and Dharki), Pakistan. The results indicate that pH, color and odor were all within limits of WHO that is pH ranges 6.5–8.5, colorless and odorless, respectively. The high values of suspended solids were observed in the Sindh-1 and Dharki samples. Microbiologically only Sahiwal and Jawar Dharki were found fit for drinking purpose. Trace metals analysis of Sheikhupura-1 and Sindh-1 showed that values do not fall within limits of WHO for Iron. The ionic concentration analysis showed that high bicarbonate (HCO3-), ions are present in the samples of Sahiwal and Dharki; Sindh-1 and Jawar Dharki samples showed very high concentration for chloride ions, all samples were satisfactory level for sulphate (SO42-), sodium, magnesium and phosphate ions except samples of Sindh-1 and Jawar Dharki. High concentration of calcium and potassium ions was observed in samples of Sindh-1, while all other samples were found fit for drinking purposes in respect of nitrate, nitrite and ammonium ions. The high concentration of Fluoride was found only in Sheikhupura-2 samples.

Investigation of the effect of loratadine and calcium ions on oxidoreductase activity of catalase enzyme

2020 ◽  
Vol 16 ◽  
Author(s):  
Edhem Hasković ◽  
Safija Herenda ◽  
Zehra Halilović ◽  
Snežana Unčanin ◽  
Denis Hasković ◽  
...  
Keyword(s):  
Calcium Ions ◽  
Enzyme Reaction ◽  
Analytical Techniques ◽  
Competitive Inhibitor ◽  
Cellular Functions ◽  
Oxidoreductase Activity ◽  
H1 Antihistamines ◽  
Activity Of Enzymes ◽  
The Given

Background: The Spectrophotometric method is one of the most suitable analytical techniques for testing the activity of enzymes under the influence of various factors. Methods: The effect of H1-antihistamines of loratadine and calcium ions on enzyme catalase under in vitro conditions was investigated in this paper. Results and Discussion: It has been shown that loratadine isa partial inhibitor of catalase, but this effect is diminished in the presence of calcium ions. Calcium as well as other cations are important for many biological and cellular functions. The kidneys play a central role in the homeostasis of these ions. The activity of the catalase enzyme under the given conditions, the type of inhibition,and the kinetic parameters of the enzyme reaction were determined. Conclusion: We concluded that loratadine is a partially competitive inhibitor.

scholarly journals Human GBP1 differentially targets Salmonella and Toxoplasma to license recognition of microbial ligands and caspase-mediated death

2019 ◽  
Author(s):  
Daniel Fisch ◽  
Barbara Clough ◽  
Marie-Charlotte Domart ◽  
Vesela Encheva ◽  
Hironori Bando ◽  
...  
Keyword(s):  
Cell Death ◽  
Feedback Inhibition ◽  
Human Macrophages ◽  
Bacterial Surface ◽  
Caspase 1 ◽  
Host Cell Death ◽  
Cell Assays ◽  
A Site ◽  
Detection Mechanisms ◽  
Immune Detection

Guanylate binding proteins (GBPs), a family of interferon (IFN)-inducible GTPases, can promote cell-intrinsic defense by removal of intracellular microbial replicative niches through host cell death. GBPs target pathogen-containing vacuoles or the pathogen itself, and assist in membrane-disruption and release of microbial molecules that trigger cell death by activating the inflammasomes. We previously showed that GBP1 mediates atypical apoptosis or pyroptosis of human macrophages infected with Toxoplasma gondii (Tg) or Salmonella enterica Typhimurium (STm), respectively. In mice, the p47 Immunity-related GTPases (IRGs) control the recruitment of GBPs to microbe-containing vacuoles and subsequent cell death. However, humans are devoid of functional IRGs, and the pathogen-proximal immune detection mechanisms by GBP1 are poorly understood. Here, we describe two novel single-cell assays which show that GBP1 promotes the lysis of Tg-containing vacuoles and Tg plasma membrane, resulting in the cytosolic detection of Tg-DNA. In contrast, we show GBP1 only targets cytosolic STm and does not contribute to bacterial escape into the cytosol of human macrophages. GBP1 interacts with caspase-4 and recruits it directly to the bacterial surface, where caspase-4 can be activated by LPS. During STm infection, caspase-1 cleaves and inactivates GBP1 at Asp192, a site conserved in related mammalian GBP1 proteins but not in murine Gbps. STm-infected human macrophages expressing a cleavage-deficient GBP1 mutant exhibit higher pyroptosis due to the absence of caspase-1-mediated feedback inhibition of the GBP1-caspase-4 pathway. Our comparative studies elucidate microbe-specific spatiotemporal roles of GBP1 in detecting infection and the assembly and regulation of divergent caspase signaling platforms.

A special halide-stabilizing pair and enantioselectivity mechanism of DatA

2014 ◽  
Vol 70 (a1) ◽  
pp. C450-C450
Author(s):  
Lijun Guan ◽  
Hideya Yabuki ◽  
Masahiko Okai ◽  
Jun Ohtsuka ◽  
Masaru Tanokura
Keyword(s):  
Reaction Mechanism ◽  
Crystal Structures ◽  
Structural Information ◽  
Docking Simulation ◽  
Competitive Inhibitor ◽  
Residue Pair ◽  
Structural Basis ◽  
Chiral Compounds ◽  
In The Wild ◽  
Agrobacterium Tumefaciens C58

A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. DatA possesses a unique Asn-Tyr residue pair instead of the Asn-Trp residue pair conserved among the subfamily members, thus the structural basis for its reaction mechanism merits elucidation. In addition, DatA is potentially useful for pharmaceutical and environmental applications, though several crystal structures of HLD-II dehalogenases have been reported so far, the determination of the DatA structure will provide an important contribution to those fields. This work provided insight into the reaction mechanism of DatA via a combination of X-ray crystallographic and computational analysis. The crystal structures of DatA and the Y109W mutant were determined at 1.70 Å [1] and 1.95 Å, respectively. The location of the active site was confirmed by using its novel competitive inhibitor, CHES. The structural information from these two crystal structures and the docking simulation with 1,3-dibromopropane revealed that the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for 1,3-dibromopropane, due to the extra hydrogen bond between Trp109 and halogenated compounds; and that the key residue to bind halogenated substrate is only Asn43 in the wild-type DatA, while those in the Y109W mutant are the Asn-Trp pair. Furthermore, docking simulation using the crystal structures of DatA and some chiral compounds indicated that enantioselectivity of DatA toward brominated alkanes is determined by the large and small spaces around the halogen binding site.

Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

1989 ◽  
Vol 152 (1) ◽  
pp. 58-63 ◽  
Author(s):  
Edward T. Buurman ◽  
Jill Pennock ◽  
David W. Tempest ◽  
M. Joost Teixeira de Mattos ◽  
Oense M. Neijssel
Keyword(s):  
Chemostat Culture ◽  
Potassium Ions ◽  
Ammonium Ions ◽  
Micro Organisms

scholarly journals Corticosterone Can Act at the Posterior Paraventricular Thalamus to Inhibit Hypothalamic-Pituitary-Adrenal Activity in Animals that Habituate to Repeated Stress

Endocrinology ◽  
2006 ◽  
Vol 147 (10) ◽  
pp. 4917-4930 ◽  
Author(s):  
Azra Jaferi ◽  
Seema Bhatnagar
Keyword(s):  
Negative Feedback ◽  
Stress Responses ◽  
Basolateral Amygdala ◽  
Acute Stress ◽  
Feedback Inhibition ◽  
Hypothalamic Pituitary Adrenal ◽  
Repeated Stress ◽  
Paraventricular Thalamus ◽  
A Site ◽  
Time Frames

Glucocorticoids released by stress bind to glucocorticoid (GR) and/or mineralocorticoid receptors (MR) to exert negative feedback of subsequent hypothalamic-pituitary-adrenal (HPA) responses to stress. Feedback inhibition is implicated in habituation of HPA activity to repeated exposure to the same (homotypic) stressor. We hypothesized that the posterior paraventricular thalamus (pPVTh) is a site where corticosterone acts to exert negative feedback during repeated stress and that is important for habituation. As previously reported, the pPVTh inhibits HPA responses to homotypic and heterotypic stressors in repeatedly, but not acutely, stressed rats. We conducted a series of experiments involving intra-pPVTh administration of MR and/or GR agonists or antagonists during different time frames over 8 d of restraint. MR exist in the pPVTh, as do GR as shown by our immunocytochemical results. Acute intra-pPVTh injection of MR and/or GR antagonist before the eighth restraint did not alter expression of habituation. Because habituation may develop before d 8, we manipulated GR and MR in the pPVTh throughout 8 d of stress using intra-pPVTh corticosterone implants, which enhanced habituation on d 8 without affecting acute stress responses. Conversely, daily intra-pPVTh injections of GR and MR antagonists on d 1–7 of restraint prevented habituation on d 8. These data suggest that corticosterone released during repeated stress can act at GR and MR in the pPVTh to inhibit HPA responses to homotypic stress. We also found that some GR-containing cells in the pPVTh project to the medial prefrontal cortex and basolateral amygdala, suggesting that pPVTh-induced inhibition of HPA activity is potentially mediated by its projections to these select limbic structures.

scholarly journals Effects of the transport site conformation on the binding of external NAP-taurine to the human erythrocyte anion exchange system. Evidence for intrinsic asymmetry.

1984 ◽  
Vol 83 (5) ◽  
pp. 683-701 ◽  
Author(s):  
P A Knauf ◽  
F Y Law ◽  
T Tarshis ◽  
W Furuya
Keyword(s):  
Anion Exchange ◽  
Chloride Transport ◽  
Chloride Concentration ◽  
Competitive Inhibitor ◽  
Disulfonic Acid ◽  
Inhibitory Potency ◽  
Intracellular Chloride ◽  
Chloride Exchange ◽  
A Site ◽  
Transport Site

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell chloride exchange by binding to a site that is distinct from the chloride transport site. Increases in the intracellular chloride concentration (at constant external chloride) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a chloride gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular chloride on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo ) form. Since NAP-taurine prevents the conformational change from EClo to ECli , it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4'-diisothiocyano-1,2- diphenylethane -2,2'-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of chloride gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Cli = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.

Sign in / Sign up

Export Citation Format

Share Document