scholarly journals Inactivation kinetics of dihydrofolate reductase from Chinese hamster during urea denaturation

1997 ◽  
Vol 324 (2) ◽  
pp. 395-401 ◽  
Author(s):  
Jia-Wei WU ◽  
Zhi-Xin WANG ◽  
Jun-Mei ZHOU
Keyword(s):  
Enzyme Activity ◽  
Dihydrofolate Reductase ◽  
Single Phase ◽  
Tertiary Structure ◽  
Chinese Hamster ◽  
Free Enzyme ◽  
Inactivation Kinetics ◽  
Binary And Ternary Complexes ◽  
Enzyme Substrate ◽  
Kinetics Of

The kinetic theory of substrate reaction during modification of enzyme activity has been applied to the study of inactivation kinetics of Chinese hamster dihydrofolate reductase by urea [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381–436]. On the basis of the kinetic equation of substrate reaction in the presence of urea, all microscopic kinetic constants for the free enzyme and enzyme–substrate binary and ternary complexes have been determined. The results of the present study indicate that the denaturation of dihydrofolate reductase by urea follows single-phase kinetics, and changes in enzyme activity and tertiary structure proceed simultaneously in the unfolding process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation, and enzyme–substrate complexes lose their activity less rapidly than the free enzyme.

scholarly journals Activation mechanism and modification kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate

1998 ◽  
Vol 335 (1) ◽  
pp. 181-189 ◽  
Author(s):  
Jia-Wei WU ◽  
Zhi-Xin WANG
Keyword(s):  
Binding Energy ◽  
Dihydrofolate Reductase ◽  
Tertiary Structure ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Activation Mechanism ◽  
Binary And Ternary Complexes ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Kinetics Of

Substrate effects on the activation kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the presence of pCMB, all modification kinetic constants for the free enzyme and enzyme–substrate binary and ternary complexes have been determined. The results of the present study indicate that the modification of Chinese hamster dihydrofolate reductase by pCMB shows single-phase kinetics, and that changes in the enzyme activity and tertiary structure proceed simultaneously during the modification process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against modification by pCMB. In the presence of a saturating concentration of NADPH, the value of kcat for 7,8-dihydrofolate in the enzyme-catalysed reaction increased four-fold on modification of Cys-6, accompanied by a two-fold increase in Km for the modified enzyme. The utilization of the binding energy of a group to increase kcat rather than reduce Km implies that the full binding energy of the group is not realized in the formation of the enzyme–substrate complex, but is used to stabilize the enzyme–transition-state complex.

Activity and conformational changes of horseradish peroxidase in trifluoroethanol

2002 ◽  
Vol 80 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Hong-Wei Zhou ◽  
Yan Xu ◽  
Hai-Meng Zhou
Keyword(s):  
Enzyme Activity ◽  
Horseradish Peroxidase ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intrinsic Fluorescence ◽  
Two Phase ◽  
Main Effect ◽  
Α Helix ◽  
Kinetics Of

The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5–25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25–40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The α-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.Key words: horseradish peroxidase, trifluoroethanol, unfolding, Soret.

scholarly journals The effect of pH on the kinetics of arylsulphatases A and B

1983 ◽  
Vol 213 (3) ◽  
pp. 603-607 ◽  
Author(s):  
C O'Fagain ◽  
B M Butler ◽  
T J Mantle
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Substrate Binding ◽  
Acid Base ◽  
Effect Of Ph ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Kinetics Of

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.

Thermal Inactivation at High Temperatures and Regeneration of Green Asparagus Peroxidase

1996 ◽  
Vol 59 (10) ◽  
pp. 1065-1071 ◽  
Author(s):  
CARMEN RODRIGO ◽  
MIGUEL RODRIGO ◽  
ANDRÉS ALVARRUIZ ◽  
ANA FRÍGOLA
Keyword(s):  
Enzyme Activity ◽  
Peroxidase Activity ◽  
Thermal Inactivation ◽  
Maximum Activity ◽  
Small Samples ◽  
Inactivation Kinetics ◽  
Optimum Conditions ◽  
First Order ◽  
Kinetics Of ◽  
Green Asparagus

A spectrophotometric method was developed for determining the peroxidase activity of green asparagus in small samples. The optimum conditions for the analysis in the cuvette were 45 mM of H2O2 36 mM of guaiacol, and pH 7. The method can be used to determine enzyme activity at up to two decimal reductions. A study was performed of the regeneration and inactivation kinetics of the enzyme when heated between 90 and 125°C. Regenerated asparagus peroxidase reached its maximum activity after being stored 6 days at 25°C. The regenerated enzyme followed first-order inactivation kinetics, showing an Ea = 13.62 kcal/mol and k100°C = 2.07 min−1.

scholarly journals Kinetics of inactivation of bovine pancreatic ribonuclease A by bromopyruvic acid

1996 ◽  
Vol 320 (1) ◽  
pp. 187-192 ◽  
Author(s):  
Ming-Hua WANG ◽  
Zhi-Xin WANG ◽  
Kang-Yuan ZHAO
Keyword(s):  
Competitive Inhibition ◽  
Ribonuclease A ◽  
Inactivation Kinetics ◽  
Ionization State ◽  
Substrate Complex ◽  
Irreversible Inhibition ◽  
Enzyme Product ◽  
Enzyme Substrate ◽  
Pancreatic Ribonuclease A ◽  
Kinetics Of

The kinetic theory of substrate reaction during the modification of enzyme activity [Duggleby (1986) J. Theor. Biol. 123, 67–80; Wang and Tsou (1990) J. Theor. Biol. 142, 531–549] has been applied to a study of the inactivation kinetics of ribonuclease A by bromopyruvic acid. The results show that irreversible inhibition belongs to a non-competitive complexing type inhibition. On the basis of the kinetic equation of substrate reaction in the presence of the inhibitor, all microscopic kinetic constants for the free enzyme, the enzyme–substrate complex and the enzyme–product complex have been determined. The non-competitive inhibition type indicates that neither the substrate nor the product affects the binding of bromopyruvic acid to the enzyme and that the ionization state of His-119 may be the same in both the enzyme–substrate and the enzyme–product complexes.

scholarly journals Kinetics of the course of inactivation of aminoacylase by 1,10-phenanthroline

1992 ◽  
Vol 281 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Z X Wang ◽  
H B Wu ◽  
X C Wang ◽  
H M Zhou ◽  
C L Tsou
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Inactivation Kinetics ◽  
Slow Inactivation ◽  
Phenanthroline Complex ◽  
Rapid Reaction ◽  
Kinetics Of ◽  
Active Site Conformation ◽  
Inhibited Enzyme ◽  
Substrate Competition

The kinetic theory of the substrate reaction during modification of enzyme activity previously described [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436] has been applied to a study on the kinetics of the course of inactivation of aminoacylase by 1,10-phenanthroline. Upon dilution of the enzyme that had been incubated with 1,10-phenanthroline into the reaction mixture, the activity of the inhibited enzyme gradually increased, indicating dissociation of a reversible enzyme–1,10-phenanthroline complex. The kinetics of the substrate reaction with different concentrations of the substrate chloroacetyl-L-alanine and the inactivator suggest a complexing mechanism for inactivation by, and substrate competition with, 1,10-phenanthroline at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-Zn(2+)-1,10-phenanthroline complex is a relatively rapid reaction, followed by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity.

scholarly journals Temperature and enzyme activity in poikilotherms. Isocitrate dehydrogenases in rainbow-trout liver

1971 ◽  
Vol 123 (5) ◽  
pp. 695-705 ◽  
Author(s):  
Thomas W. Moon ◽  
P. W. Hochachka
Keyword(s):  
Enzyme Activity ◽  
Rainbow Trout ◽  
Isocitrate Dehydrogenase ◽  
Low Temperatures ◽  
Substrate Affinity ◽  
Thermally Induced ◽  
Cold Adapted ◽  
Enzyme Substrate ◽  
Kinetics Of ◽  
Rainbow Trout Liver

1. The kinetics of the thermally induced enzyme variants of the supernatant NADP–isocitrate dehydrogenase from rainbow-trout liver are investigated. 2. Fish acclimatized to 2°C (cold-adapted enzyme) and 17°C (warm-adapted enzyme) show different relative distributions of the three NADP–isocitrate dehydrogenase isoenzymes; this has been demonstrated with electrophoresis and electrofocusing techniques. 3. Plots of Km versus temperature for the cold-adapted and warm-adapted enzyme variants are complex in nature with apparent maximal enzyme–substrate affinity corresponding to the temperature at which the trout is acclimatized. Both substrates, dl-isocitrate and NADP+, give similar curves although the magnitude of the Km change with temperature is much decreased in the case of NADP+. 4. Ea values of approx. 18kcal/mol were determined for both the cold-adapted and warm-adapted enzyme variants. 5. In an attempt to determine how velocities can be increased at low temperatures, cation, pH requirements, metabolite and enzyme concentrations were examined. 6. NAD–isocitrate dehydrogenase could not be detected in trout tissues.

Analysis of process of emulsions transport in hydrophilic/oleophilic granular porous media driven by capillary force

2018 ◽  
Vol 2 (21) ◽  
pp. 85-101
Author(s):  
Olga Shtyka ◽  
Łukasz Przybysz ◽  
Mariola Błaszczyk ◽  
Jerzy P. Sęk
Keyword(s):  
Porous Media ◽  
Experimental Research ◽  
Granular Media ◽  
Single Phase ◽  
Influential Factor ◽  
Dispersed Phase ◽  
Porous Structures ◽  
Phase Concentration ◽  
Granular Porous Media ◽  
Kinetics Of

The research focuses on the issues concerning a process of multiphase liquids transport in granular porous media driven by the capillary pressure. The current publication is meant to introduce the results of experimental research conducted to evaluate the kinetics of the imbibition and emulsions behavior inside the porous structures. Moreover, the influence of the dispersed phase concentration and granular media structure on the mentioned process was considered. The medium imbibition with emulsifier-stabilized emulsions composed of oil as the dispersed phase in concentrations of 10 vol%, 30 vol%, and 50 vol%, was investigated. The porous media consisted of oleophilic/hydrophilic beads with a fraction of 200–300 and 600–800 μm. The experimental results provided that the emulsions imbibition in such media depended stronger on its structure compare to single-phase liquids. The increase of the dispersed phase concentration caused an insignificant mass decreasing of the imbibed emulsions and height of its penetration in a sorptive medium. The concentrations of the imbibed dispersions exceeded their initial values, but reduced with permeants front raise in the granular structures that can be defined as the influential factor for wicking process kinetics.

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