scholarly journals Control of the flux in the arginine pathway of Neurospora crassa. Modulations of enzyme activity and concentration

1981 ◽  
Vol 200 (2) ◽  
pp. 231-246 ◽  
Author(s):  
H J Flint ◽  
R W Tateson ◽  
I B Barthelmess ◽  
D J Porteous ◽  
W D Donachie ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Genetic Recombination ◽  
Quantitative Measure ◽  
Sensitivity Coefficient ◽  
Mass Action ◽  
Rate Limiting Step ◽  
One Step ◽  
Rate Limiting ◽  
Different Parts

The influence of particular enzyme activities on the flux of metabolites in a pathway can be estimated by ‘modulating’ enzymes (i.e. changing turnover or concentration) and measuring the response in various parts of the system. By controlling the nuclear ration of two genetically different nuclear types in heterokaryons, the enzyme concentrations at four different steps in the arginine pathway were decreased over a range. This range was extended by the use of bradytrophs, mutant strains specifying enzymes with greatly diminished enzyme activities. Strains altered simultaneously at more than one step were also constructed by genetic recombination. By measuring the outputs of the pathway and the steady-state concentrations of intermediate pools, the fluxes in different parts of the pathway were calculated. This allowed the construction of flux/enzyme relationships, the slope of which is a measure of the sensitivity of a flux to the change in enzyme activity at that step. All fluxes were found to be considerably buffered for quite substantial decreases in the activities of all enzymes. Mass action plays an important part in this phenomenon, as do inhibition and repression. Because of the existence of expansion fluxes in growing systems, we find quantitatively different fluxes in different parts of the single pathway. For the same reason some enzyme modulations given decreased fluxes in one part and increased fluxes in another. The understanding of control in the pathway thus involves consideration of many mechanisms operating simultaneously and the estimation of changes in the whole system. The concept of a ‘rate-limiting step’ is found to be inadequate and is replaced by a quantitative measure, the Sensitivity Coefficient, which takes account of all the interactions. It is shown that control of the flux is shared among all the enzymes of the pathway. The results are discussed in terms of the theory of flux control.

scholarly journals New Spectrophotometric Method for the Assessment of Catalase Enzyme Activity in Biological Tissues

2020 ◽  
Vol 16 (8) ◽  
pp. 1054-1062 ◽  
Author(s):  
Thulfeqar A. Hamza ◽  
Mahmoud H. Hadwan
Keyword(s):  
Hydrogen Peroxide ◽  
Enzyme Activity ◽  
Clinical Pathology ◽  
Biological Tissues ◽  
Ammonium Molybdate ◽  
Spectrophotometric Methods ◽  
Rate Limiting Step ◽  
Coefficients Of Variation ◽  
Rate Limiting ◽  
Health Applications

Background: Catalase is a vital antioxidant enzyme that dismutates H2O2 into water and molecular oxygen. Many protocols have been developed to measure catalase enzyme activity. Spectrophotometric methods are the most common assays that used to assess catalase enzyme activity. Methods: Because the rate-limiting step during catalase enzyme activity depends upon the dissociation of hydrogen peroxide, the developed assay measures the reaction between a hydroquinone/ anilinium sulfate/ammonium molybdate reagent and Unreacted Hydrogen Peroxide, which results in the production of a purple, disubstituted quinone compound with a maximum absorbance value at 550 nm. Results: To clarify the precision of the developed method, the coefficients of variation were determined to be 2.6% and 4.7% within run measurements and between run measurements, respectively. This method returned results that correlated well (r = 0.9982) with the results returned using the peroxovanadate method to assess catalase enzyme activity. Additionally, we examined the use of the newly developed hydroquinone assay to measure catalase enzyme activity in liver and bacterial homogenate samples. Conclusion: These results demonstrated that this assay can be used for scientific research and routine health applications because it is inexpensive, simple, accurate, and rapid. This method is suitable for use in clinical pathology laboratories because it is simple and produces precise and reproducible results.

Determination of the Isotope Effect of the Enzymatic Oxidation of (R)Carnitine by Displacement of the Equilibrium via Mass Action

1975 ◽  
Vol 30 (7-8) ◽  
pp. 438-441
Author(s):  
Klaus Brendel ◽  
Rubin Bressler ◽  
Miguel A. Alizade
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Isotope Effect ◽  
Mass Action ◽  
Enzymatic Oxidation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Different Temperatures ◽  
Acid Hydrochloride ◽  
Rate Limiting

Abstract An isotope effect of the dehydrogenation of (R) Carnitine [(R) 3-hydroxy-4-trimethylamino-butyric acid hydrochloride] catalyzed by (R) carnitine dehydrogenase [(R) carnitine: NAD oxido-reductase E.C. 1.1.1.108] from Pseudomonas aeruginosa was measured at different temperatures. It was found that k1H/k3H does not vary greatly with changes of temperature. The value of 3 for k1H/k3H measured at small initial conversions strongly indicated that the rate limiting step of the oxidation of (R) carnitine is the cleavage of the C-H bond at C3.

scholarly journals Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king-crab

1971 ◽  
Vol 121 (3) ◽  
pp. 399-409 ◽  
Author(s):  
Hans W. Behrisch
Keyword(s):  
Skeletal Muscle ◽  
Enzyme Activity ◽  
Low Temperature ◽  
Temperature Sensitive ◽  
King Crab ◽  
Rate Limiting Step ◽  
Low Concentrations ◽  
Higher Temperature ◽  
Paralithodes Camtschatica ◽  
Rate Limiting

1. The properties of fructose diphosphatase from skeletal muscle of the Alaskan king-crab (Paralithodes camtschatica) were examined over the physiological temperature range of the animal. 2. King-crab muscle fructose diphosphatase is first activated by Na+ and NH4+ and is then partially inhibited by these cations at concentrations higher than 10mm at 0°, 8° and 15°C. Enzyme activity is stimulated by K+ at 0°C, but is curtailed at 8°C and 15°C, an effect that could render rate independent of temperature. 3. Affinity for substrate increases with decreasing temperature; below the temperature of acclimatization, Km for fructose 1,6-diphosphate increases, resulting in a complex U-shaped temperature–Km curve. 4. King-crab muscle fructose diphosphatase is inhibited by low concentrations of AMP. As with enzymes of other poikilotherms, inhibition by AMP is sensitive to temperature; the enzyme is least sensitive to inhibition by AMP near the temperature of acclimatization. 5. The affinity of fructose diphosphatase for fructose 1,6-diphosphate is enhanced by phosphoenolpyruvate, and this activation is temperature-sensitive; 0.5mm-phosphoenolpyruvate causes a sevenfold decrease in Km for fructose 1,6-diphosphate at 15°C but a 25-fold decrease at 0°C. 6. Phosphoenolpyruvate appears to decrease the affinity of king-crab muscle fructose diphosphatase for AMP at low temperature, whereas at the higher temperature it appears to enhance inhibition by AMP. Phosphoenolpyruvate was not observed to cause a reversal of inhibition of fructose diphosphatase activity by AMP. The identification of phosphoenolpyruvate as an activator of a rate-limiting step in gluconeogenesis permits the suggestion of a coupling of the controlling mechanisms of several steps in the glycolytic and gluconeogenic chains. 7. These findings suggest mechanisms for the maintenance and regulation of control of fructose diphosphatase activity in king-crab skeletal muscle at low temperature and under conditions that favour concomitant activity of phosphofructokinase.

scholarly journals Enzyme activity and dynamics: xylanase activity in the absence of fast anharmonic dynamics

2000 ◽  
Vol 346 (2) ◽  
pp. 355-358 ◽  
Author(s):  
Rachel V. DUNN ◽  
Valerie RÉAT ◽  
John FINNEY ◽  
Michel FERRAND ◽  
Jeremy C. SMITH ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thermotoga Maritima ◽  
Xylanase Activity ◽  
Sharp Transition ◽  
Temperature Range ◽  
Internal Motions ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Single Subunit

The activity and dynamics of a simple, single subunit enzyme, the xylanase from Thermotoga maritima strain Fj SS3B.1 have been measured under similar conditions, from -70 to +10 °C. The internal motions of the enzyme, as evidenced by neutron scattering, undergo a sharp transition within this temperature range; they show no evidence for picosecond-timescale anharmonic behaviour (e.g. local diffusive motions or jumps between alternative conformations) at temperatures below -50 °C, whereas these motions are strongly activated at higher temperatures. The activity follows Arrhenius behaviour over the whole of the temperature range investigated, -70 to +10 °C. The results indicate that a temperature range exists over which the enzyme rate-limiting step is independent of fast anharmonic dynamics.

scholarly journals New Spectrophotometric Method for the Assessment of Catalase Enzyme Activity in Biological Tissues

2019 ◽  
Author(s):  
Thulfeqar A. Hamza ◽  
Mahmoud Hussein Hadwan
Keyword(s):  
Hydrogen Peroxide ◽  
Enzyme Activity ◽  
Clinical Pathology ◽  
Biological Tissues ◽  
Ammonium Molybdate ◽  
Spectrophotometric Methods ◽  
Rate Limiting Step ◽  
Coefficients Of Variation ◽  
Rate Limiting ◽  
Health Applications

Background: Catalase is a vital antioxidant enzyme that dismutates H2O2 into water and molecular oxygen. Many protocols have been developed to measure catalase enzyme activity. Spectrophotometric methods are the most common assays that used to assess catalase enzyme activity. Methods: Because the rate-limiting step during catalase enzyme activity depends upon the dissociation of hydrogen peroxide, the developed assay measures the reaction between a hydroquinone/anilinium sulfate/ammonium molybdate reagent and Unreacted Hydrogen Peroxide, which results in the production of a purple, disubstituted quinone compound with a maximum absorbance value at 550 nm. Results: To clarify the precision of the developed method, the coefficients of variation were determined to be 2.6% and 4.7% for within run measurements and between run measurements, respectively. This method returned results that correlated well (r = 0.9982) with the results returned using the peroxovanadate method to assess catalase enzyme activity. Additionally, we examined the use of the newly developed hydroquinone assay to measure catalase enzyme activity in liver and bacterial homogenate samples. Conclusion: These results demonstrated that this assay can be used for scientific research and routine health applications because it is inexpensive, simple, accurate, and rapid. This method is suitable for use in clinical pathology laboratories because it is simple and produces precise and reproducible results.

A modified capillary suction time apparatus for measuring the filterability of super-flocculated sludges

2000 ◽  
Vol 42 (9) ◽  
pp. 135-139 ◽  
Author(s):  
P. A. Vesilind ◽  
B. Örmeci
Keyword(s):  
Filter Paper ◽  
Free Water ◽  
Quantitative Measure ◽  
Water Release ◽  
Capillary Suction ◽  
Freeze Thaw ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
To Come ◽  
Rate Limiting

The capillary suction time (CST) test provides a quantitative measure of the rate of water release from a sludge and has the advantage of being simple to run. The test consists of allowing sludge water to filter into and penetrate a filter paper by capillary force. Although the CST works well for the majority of sludges, it is not a useful test for measuring the filterability of highly flocculated sludges or sludges conditioned by other methods such as freeze-thaw. The escape of water from the sludge in such cases is too fast and the rate-limiting step becomes the movement of water through the filter paper instead of the release of water from the sludge. There is a need to improve the CST apparatus so it can be used to measure the degree of filterability for super flocculated sludges as well. Such a device is described in this paper. It consists of first draining the free water from the well-flocculated sludge and only then allowing the flocculated sludge to come in contact with the filter paper.The results of our study show that the filterability of super-flocculated sludges can be measured with acceptable precision using the modified CST apparatus.

scholarly journals Transient and intermediate carbocations in ruthenium tetroxide oxidation of saturated rings

2019 ◽  
Vol 15 ◽  
pp. 1552-1562
Author(s):  
Manuel Pedrón ◽  
Laura Legnani ◽  
Maria-Assunta Chiacchio ◽  
Pierluigi Caramella ◽  
Tomás Tejero ◽  
...  
Keyword(s):  
Topological Analysis ◽  
Ruthenium Tetroxide ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Iminium Ion ◽  
Single Transition ◽  
One Step ◽  
Two Stages ◽  
Rate Limiting ◽  
Mediated Oxidation

The ruthenium tetroxide-mediated oxidation of cyclopentane, tetrahydrofuran, tetrahydrothiophene andN-substituted pyrrolidines has been studied computationally by DFT and topological (analysis of the electron localization function, ELF) methods. In agreement with experimental observations and previous DFT calculations, the rate-limiting step of the reaction takes place through a highly asynchronous (3 + 2) concerted cycloaddition through a single transition structure (one kinetic step). The ELF analysis identifies the reaction as a typical one-step-two-stages process and corroborates the existence of a transient carbocation. In the case of pyrrolidines, the carbocation is completely stabilized as an energy minimum in the form of an iminium ion and the reaction takes place in two steps.

Solvolysis kinetics and mechanism of 3-methyl-1,3-thiazolidine-2,4-dione

1981 ◽  
Vol 46 (12) ◽  
pp. 3097-3103 ◽  
Author(s):  
Vladimír Macháček ◽  
Vojeslav Štěrba ◽  
Helena Zahradníčková
Keyword(s):  
Sodium Methoxide ◽  
Kinetics And Mechanism ◽  
Hydrolysis Kinetics ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Reaction Proceeds ◽  
One Step ◽  
Rate Limiting ◽  
Kinetics Of

The hydrolysis kinetics of 3-methyl-1,3-thiazolidine-2,4-dione have been studied in aqueous buffers and dilute NaOH solutions. The reaction proceeds via two base-catalyzed steps having different rates. In sodium methoxide solutions 3-methyl-1,3-thiazolidine-2,4-dione undergoes one-step methanolysis giving methyl thioglycolate anion as the final product. The rate-limiting step consists in decomposition of the anion CH3NCOSCH2COOCH3.

scholarly journals One-Step Biosynthesis of Vitamin C in Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Mengyu Zhou ◽  
Yanhui Bi ◽  
Mingzhu Ding ◽  
Yingjin Yuan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vitamin C ◽  
Clinical Medicine ◽  
Rate Limiting Step ◽  
Exogenous Addition ◽  
One Step ◽  
Galactose Dehydrogenase ◽  
First Time ◽  
Rate Limiting

Vitamin C (VC) is comprehensively applied in foods, cosmetics, pharmaceuticals, and especially clinical medicine. Nowadays, the industrial production of VC mainly relies on the classic two-step fermentation route, and researchers have explored the way for one-step fermentation of VC in recent years. In this study, a VC biosynthesis pathway that directly produced VC from glucose was reconstructed in Saccharomyces cerevisiae, and the protein engineering and metabolic engineering strategies were adopted to improve it. First, five exogenous modules from Arabidopsis were introduced into the chassis cells by synthetic biology approaches to obtain the strain YLAA harboring VC biosynthesis. In addition, L-galactose dehydrogenase (L-GalDH) and L-galactono-1,4-lactone dehydrogenase (L-GLDH) were fused and expressed in S. cerevisiae cells for the first time, which increased the intracellular VC accumulation by 2.78-fold, reaching 9.97 ± 0.09 mg/L. Through copy number engineering, it was further confirmed that the last step catalyzed by L-GLDH is the rate-limiting step. GDP-L-galactose phosphorylase (GPP) encoded by vtc2 is another rate-limiting enzyme confirmed by GAL1p overexpression results. Finally, by balancing gene expression and cell growth, the highest production strain with overexpressing vtc2 by multicopy plasmids was constructed. The VC accumulation reached 24.94 ± 1.16 mg/L, which was currently the highest production from glucose in S. cerevisiae. The production of the recombinant strain reached nearly 44 mg/L with the exogenous addition of L-galactose or glutathione. The results further emphasized the importance of the step catalyzed by GPP. The investigation provided experience for the efficient biosynthesis of VC and the determination of rate-limiting steps.

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