scholarly journals Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding

1996 ◽  
Vol 314 (3) ◽  
pp. 985-991 ◽  
Author(s):  
Subrata ADAK ◽  
Abhijit MAZUMDER ◽  
Ranajit K. BANERJEE
Keyword(s):  
Active Site ◽  
Native Enzyme ◽  
Tyrosine Residue ◽  
Tyrosine Residues ◽  
First Order ◽  
First Order Kinetics ◽  
Aromatic Donor ◽  
Pseudo First Order ◽  
Modified Enzyme ◽  
Compound Ii

The plausible role of arginine and tyrosine residues at the active site of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate constants of 24 M-1·min-1, 0.8 M-1·min-1 and 0.54 M-1·min-1 respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0 M-1·min-1. The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement of arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (∆G´) for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete inactivation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35 mM compared with 10 mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichroism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.

Active-site-directed inactivation of Schizophyllum commune cellulase by 4′, 5′-epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside

1988 ◽  
Vol 66 (8) ◽  
pp. 871-879 ◽  
Author(s):  
Anthony John Clarke
Keyword(s):  
Active Site ◽  
Schizophyllum Commune ◽  
Competitive Inhibitor ◽  
Irreversible Inhibitor ◽  
Inhibitor Molecule ◽  
Enzyme Active Site ◽  
First Order ◽  
First Order Kinetics ◽  
Pseudo First Order ◽  
Inactivation Process

4′,5′-Epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside (4) was synthesized by a Koenigs–Knorr reaction of 4-penten-1-ol and acetobromcellobiose, promoted by silver trifluoromethanesulfonate and N,N′-tetramethylurea, and tested as a potential active-site-directed irreversible inhibitor of the Schizophyllum commune cellulase. Incubation of the S. commune cellulase with 4 resulted in a time-dependent irreversible inactivation of the enzyme. The inactivation process obeyed pseudo-first-order kinetics and the hyperbolic plot of kobs as a function of inhibitor concentration provided values for Kd and k2 of 150 mM and 2.0 × 10−4 s−1, respectively, at pH 5.5 and 25 °C. The binding of a competitive inhibitor, cellobiose, to the cellulase prior to incubation with 4 protected the enzyme from rapid inactivation, suggesting that the inactivation is due to attack at the active site. The dependence of the inactivation on pH is consistent with the participation of carboxyl groups. Treatment of the affinity-labeled enzyme with [14C]methoxyamine resulted in the near stoichiometric formation of a stable radiolabelled adduct, suggesting that one inhibitor molecule binds per enzyme active site of the enzyme.

scholarly journals Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver

1981 ◽  
Vol 195 (3) ◽  
pp. 735-743 ◽  
Author(s):  
S Ramakrishna ◽  
W B Benjamin
Keyword(s):  
Rat Liver ◽  
Active Site ◽  
Arginine Residue ◽  
Atp Citrate Lyase ◽  
First Order ◽  
Complete Inactivation ◽  
First Order Kinetics ◽  
Citrate Lyase ◽  
Arginine Residues ◽  
Pseudo First Order

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.

In-vitro Kinetic Hydrolysis Study of Metronidazole Derivatives with Carvacrol and Eugenol Using Validated RP-HPLC Method

2020 ◽  
Vol 16 ◽  
Author(s):  
M. Alarjah
Keyword(s):  
Half Life ◽  
Hplc Method ◽  
Hydrolysis Rate ◽  
First Order ◽  
First Order Kinetics ◽  
Rp Hplc ◽  
Pharmacokinetic Properties ◽  
Pseudo First Order ◽  
Kinetic Hydrolysis

Background: Prodrugs principle is widely used to improve the pharmacological and pharmacokinetic properties of some active drugs. Much effort was made to develop metronidazole prodrugs to enhance antibacterial activity and or to improve pharmacokinetic properties of the molecule or to lower the adverse effects of metronidazole. Objective: In this work, the pharmacokinetic properties of some of monoterpenes and eugenol pro metronidazole molecules that were developed earlier were evaluated in-vitro. The kinetic hydrolysis rate constants and half-life time estimation of the new metronidazole derivatives were calculated using the validated RP-HPLC method. Method: Chromatographic analysis was done using Zorbbax Eclipse eXtra Dense Bonding (XDB)-C18 column of dimensions (250 mm, 4.6 mm, 5 μm), at ambient column temperature. The mobile phase was a mixture of sodium dihydrogen phosphate buffer of pH 4.5 and methanol in gradient elution, at 1ml/min flow rate. The method was fully validated according to the International Council for Harmonization (ICH) guidelines. The hydrolysis process carried out in an acidic buffer pH 1.2 and in an alkaline buffer pH 7.4 in a thermostatic bath at 37ºC. Results: The results followed pseudo-first-order kinetics. All metronidazole prodrugs were stable in the acidic pH, while they were hydrolysed in the alkaline buffer within a few hours (6-8 hr). The rate constant and half-life values were calculated, and their values were found to be 0.082- 0.117 hr-1 and 5.9- 8.5 hr., respectively. Conclusion: The developed method was accurate, sensitive, and selective for the prodrugs. For most of the prodrugs, the hydrolysis followed pseudo-first-order kinetics; the method might be utilised to conduct an in-vivo study for the metronidazole derivatives with monoterpenes and eugenol.

New insights on the UV/TiO2 photocatalytic treatment of thiomersal and its 2-sulfobenzoic acid product

2021 ◽  
Vol 02 ◽  
Author(s):  
Emmanuel M. de la Fournière ◽  
Jorge M. Meichtry ◽  
Graciela S. Custo ◽  
Eduardo A. Gautier ◽  
Marta I. Litter
Keyword(s):  
Degradation Products ◽  
Acidic Ph ◽  
Cosmetic Products ◽  
Acid Product ◽  
First Order ◽  
First Order Kinetics ◽  
Photocatalytic Destruction ◽  
Pseudo First Order ◽  
Mineralization Degree ◽  
Suitable Technique

Background: Thiomersal (TM), a complex between 2-mercaptobenzoic acid (2-MBA) and ethylmercury (C2H5Hg+), is an antimicrobial preservative used in immunological, ophthalmic, cosmetic products, and vaccines. Objective: TM has been treated by UV/TiO2 photocatalysis in the presence or absence of oxygen at acidic pH. C2H5Hg+, 2-MBA, and 2-sulfobenzoic acid (2-SBA) were found as products. A 2-SBA photocatalytic treatment was undertaken to study sulfur evolution. Methods: Photocatalytic runs were performed using a UVA lamp (λmax = 352 nm), open to the air or under N2. A suspension of the corresponding TM or 2-SBA salt and TiO2 was prepared, and pH was adjusted. Suspensions were stirred in the dark for 30 min and then irradiated. TM, 2-MBA, 2-SBA, and C2H5Hg+ were quantified by HPLC, sulfur by TXRF, and the deposits on the photocatalyst were analyzed by chemical reactions. The mineralization degree was followed by TOC. Sulfate was determined using BaCl2 at 580 nm. Results: Photocatalytic destruction of TM and total C2H5Hg+ was complete under N2 and air, but TM degradation was much faster in air. The evolution of TM and the products followed a pseudo-first-order kinetics. Conclusion: TiO2-photocatalytic degradation is a suitable technique for the treatment of TM and its degradation products. In contrast to other organomercurial compounds, TM degradation is faster in the presence of O2, indicating that the oxidative mechanism is the preferred pathway. A significant TM mineralization (> 60%, NPOC and total S) was obtained. TM was more easily degraded than 2-SBA. Sulfate was the final product.

Chemical effects induced by gas–liquid jet flow

2022 ◽  
Author(s):  
Zhiliang Zhang ◽  
Jiaqi Lu ◽  
Bingqian Lv ◽  
Wei Liu ◽  
Shuyuan Shen ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Jet Flow ◽  
Reaction Process ◽  
Liquid Jet ◽  
First Order ◽  
First Order Kinetics ◽  
Chemical Effects ◽  
Pseudo First Order

The gas-liquid jet flow was proved to be capable of inducing chemical consequences which can lead to the decomposition of methylene blue (MB). The reaction process follows a pseudo-first-order kinetics....

scholarly journals Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?

1976 ◽  
Vol 153 (2) ◽  
pp. 495-497 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Schiff Base ◽  
Rate Constant ◽  
Active Site ◽  
Order Rate Constant ◽  
Lysine Residue ◽  
Prolonged Incubation ◽  
Enzyme Active Site ◽  
First Order ◽  
Substrate Analogue ◽  
Pseudo First Order

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 × 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.

scholarly journals CATION EXCHANGE BETWEEN CELLS AND PLASMA OF MAMMALIAN BLOOD

1950 ◽  
Vol 33 (6) ◽  
pp. 703-722 ◽  
Author(s):  
C. W. Sheppard ◽  
W. R. Martin
Keyword(s):  
Exchange Rate ◽  
Radioactive Isotope ◽  
Specific Activity ◽  
Compartment System ◽  
First Order ◽  
First Order Kinetics ◽  
Mammalian Blood ◽  
The Mean ◽  
Pseudo First Order

The exchange of potassium between cells and plasma of heparinized human blood has been studied in vitro using the radioactive isotope K42. The changes in cell and plasma specific activity are characteristic of a simple two-compartment system. The mean of seven determinations of the exchange rate at 38°C. is 1.8 per cent of the cellular potassium per hour. The results indicate that at 38°C. the rate is relatively insensitive to oxygenation or reduction of the hemoglobin, and to 1200 r of gamma radiation. With varying temperature the rate follows pseudo first order kinetics with a Q10 of 2.35. Below 15°C. the rate of loss of potassium exceeds the rate of uptake.

scholarly journals The role of histidine-118 of inorganic pyrophosphatase from thermophilic bacterium PS-3

1991 ◽  
Vol 278 (2) ◽  
pp. 595-599 ◽  
Author(s):  
N Hirano ◽  
T Ichiba ◽  
A Hachimori
Keyword(s):  
Thermophilic Bacterium ◽  
Complete Loss ◽  
Inorganic Pyrophosphatase ◽  
Histidine Residue ◽  
First Order ◽  
Diethyl Pyrocarbonate ◽  
Lysyl Endopeptidase ◽  
First Order Kinetics ◽  
Pseudo First Order

Treatment of the inorganic pyrophosphatase from thermophilic bacterium PS-3 with diethyl pyrocarbonate resulted in the almost complete loss of its activity, which followed pseudo-first-order kinetics. The presence of Mg2+ prevented the inactivation. Enzyme inactivated with diethyl pyrocarbonate was re-activated by hydroxylamine. The inactivation parallelled the amount of modified histidine residue, and a plot of the activity remaining against the amount of modified histidine residue suggested that the modification of one of two histidine residues totally inactivated the enzyme. The site involved was found to be located in a single lysyl endopeptidase-digest peptide derived from the ethoxy[14C]carbonylated enzyme. Amino acid analysis and sequence analysis of the peptide revealed that it comprised residues 96-119 of the inorganic pyrophosphatase from thermophilic bacterium PS-3. These results, when compared with those reported for the Escherichia coli and yeast enzymes, imply that His-118 of the inorganic pyrophosphatase from thermophilic bacterium PS-3 is located near the Mg(2+)-binding site and thus affects the binding of Mg2+.

Effects of oxidant concentration and temperature on decolorization of azo dye: comparisons of UV/Fenton and UV/Fenton-like systems

2012 ◽  
Vol 65 (11) ◽  
pp. 1970-1974 ◽  
Author(s):  
C. Y. Kuo ◽  
C. Y. Pai ◽  
C. H. Wu ◽  
M. Y. Jian
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Azo Dye ◽  
Activation Energies ◽  
First Order ◽  
First Order Kinetics ◽  
Oxidant Concentration ◽  
Decolorization Efficiency ◽  
Pseudo First Order ◽  
Reactive Red 2

This study applies photo-Fenton and photo-Fenton-like systems to decolorize C.I. Reactive Red 2 (RR2). The oxidants were H2O2 and Na2S2O8; Fe2+, Fe3+, and Co2+ were used to activate these two oxidants. The effects of oxidant concentration (0.3–2 mmol/L) and temperature (25–55 °C) on decolorization efficiency of the photo-Fenton and photo-Fenton-like systems were determined. The decolorization rate constants (k) of RR2 in the tested systems are consistent with pseudo-first-order kinetics. The rate constant increased as oxidant concentration and temperature increased. Activation energies of RR2 decolorization in the UV/H2O2/Fe2+, UV/H2O2/Fe3+, UV/Na2S2O8/Fe2+ and UV/Na2S2O8/Fe3+ systems were 32.20, 39.54, 35.54, and 51.75 kJ/mol, respectively.

Study on Phenol Oxidation Catalyzed by Persulfate

2013 ◽  
Vol 864-867 ◽  
pp. 96-100
Author(s):  
Shen Xin Li ◽  
Wei Hu ◽  
Ying Wang ◽  
Jian Zhang Li ◽  
Cheng Duan Wang
Keyword(s):  
Schiff Base ◽  
Key Words ◽  
Industrial Wastewater ◽  
Phenol Oxidation ◽  
Molar Ratio ◽  
Optimum Temperature ◽  
First Order ◽  
First Order Kinetics ◽  
Pseudo First Order ◽  
Oxidation System

The phenol oxidation with persulfate catalyzed were studied. Effects of several parameters, such as dose of oxidant, pH, temperature and UV irradiation, were investigated in detail. The results showed that the phenol oxidation by persulfate could be fitted to a pseudo-first order kinetics model. The optimum acidity of the phenol oxidation system in the paper is ca. pH 8.76, the optimum temperature which is ca.70 °C and the optimum molar ratio of persulfate to the phenol is ca.40 in the solution.The results are useful for the treatment of industrial wastewater. Key words: Phenol oxidation Schiff base manganese (III) complexes Persulfate

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