Cysteine-sensitised formation and repair of mixed disulfide in the oxidation of papain by H2O2 and OH radicals

1976 ◽  
Vol 54 (2) ◽  
pp. 242-253 ◽  
Author(s):  
Wen Shu Lin ◽  
G. Maurice Gaucher ◽  
David A. Armstrong ◽  
Manohar Lal
Keyword(s):  
Hydrogen Peroxide ◽  
Computer Program ◽  
Active Site ◽  
Proteolytic Enzyme ◽  
Oh Radicals ◽  
Sulfenic Acid ◽  
Enzyme Active Site ◽  
Mixed Disulfide ◽  
Kinetics Of ◽  
Saturated Solutions

The inactivation of the proteolytic enzyme papain by hydrogen peroxide produces a sulfenic acid by oxidation of the essential SH of cysteine 25 at the enzyme active site:[Formula: see text]The kinetics of repair of this entity by cysteine were consistent with the two reactions:[Formula: see text][Formula: see text]Reaction 4 was the faster with k4 ≥ 800 M−1 s−1, and k5 = 11.3 ± 0.5 M−1 s−1. A computer program was developed to evaluate the contributions of peroxide-inactivation and cysteine-repair when they occur simultaneously in N2O-saturated solutions in the absence of catalase. The yields predicted by this program agreed well with the inactivation caused by peroxide in irradiated systems.The effect of cysteine on the inactivation of papain by OH radicals produced by radiolysis of N2O-saturated solutions containing catalase was also investigated. Protection against permanent inactivation was much more efficient than expected on the basis of a simple competition between cysteine and papain for OH radicals, but there was a marked increase in the yield of repairable damage which was not due to hydrogen peroxide. These observations can be qualitatively accounted for by the reactions:[Formula: see text]The same rate constant was obtained for the repair of PapainCys25SSCys from this source as from the peroxide inactivation and treatment with cysteine. However, there was also evidence for additional cysteine-sensitized production of mixed disulfide and this probably occurs through reactions of CysS• radicals:[Formula: see text]

scholarly journals Impact of Hydrogen Peroxide on the UVC Photolysis of Diclofenac and Toxicity of the Phototransformation Products

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Stanisław Ledakowicz ◽  
Emilia Drozdek ◽  
Tomasz Boruta ◽  
Magdalena Foszpańczyk ◽  
Magdalena Olak-Kucharczyk ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Vibrio Fischeri ◽  
Reaction System ◽  
Oxidation Products ◽  
Oh Radicals ◽  
Direct Photolysis ◽  
Depth Analysis ◽  
Comprehensive Comparison ◽  
Kinetics Of ◽  
Very High

The aim of this study was to investigate the effect of hydrogen peroxide on the UVC photolysis of diclofenac (DCF) in aqueous solution. The experimental results confirmed very high effectivity of UVC direct photolysis of diclofenac. Moreover, it was found that H2O2/UV only slightly improved photodegradation; however, the addition of hydrogen peroxide into the reaction system affected the mechanism of DCF decomposition. Kinetics of the DCF reaction with ⋅OH radicals in the UV/H2O2 process was determined. For both processes, namely, photolysis and UV/H2O2, an in-depth analysis focused on the formation of phototransformation products of DCF (TPs) was performed. To the best of our knowledge, such comprehensive comparison of diclofenac photodegradation via UVC photolysis and UV/H2O2 has not been presented so far. Although there were no significant differences with regard to the rate of diclofenac degradation by photolysis and UV/H2O2, different oxidation products were found to be associated with the two considered processes. Furthermore, the H2O2/UV treatment increased toxicity towards Vibrio fischeri, while direct UVC photolysis had no significant effect on toxicity. The increase in toxicity can be attributed to the breakdown of DCF and formation of much more toxic TPs in the course of the H2O2/UVC process.

scholarly journals Kinetics of protein modification reactions. Plot of fractional enzyme activity versus extent of protein modification in cases where all modifiable groups are essential for enzyme activity

1984 ◽  
Vol 223 (1) ◽  
pp. 259-262 ◽  
Author(s):  
E T Rakitzis
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Protein Modification ◽  
Enzyme Molecule ◽  
Single Group ◽  
Initial Portion ◽  
Enzyme Active Site ◽  
Kinetics Of

The plot of fractional enzyme activity versus extent of protein modification, for cases where all enzyme modifiable groups of a certain kind are essential for activity, is found to be nearly independent of the number, per enzyme active site, of modifiable groups involved. Such plots usually, by a fallacious extension of the initial portion of the plot on the extent-of-modification axis, are interpreted to mean the modification of one single group per enzyme active site (or per enzyme molecule). The possible relevance of these findings to cases in the literature is discussed.

scholarly journals Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens

2000 ◽  
Vol 345 (2) ◽  
pp. 315-319 ◽  
Author(s):  
Dieter BECKER ◽  
Karin S. H. JOHNSON ◽  
Anu KOIVULA ◽  
Martin SCHÜLEIN ◽  
Michael L. SINNOTT
Keyword(s):  
Trichoderma Reesei ◽  
Active Site ◽  
Substrate Concentration ◽  
Initial Rate ◽  
Enzyme Active Site ◽  
Humicola Insolens ◽  
Cellobiohydrolase Ii ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Identical Crystal

We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.

scholarly journals Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1679
Author(s):  
Vishnu Mohan ◽  
Jean P. Gaffney ◽  
Inna Solomonov ◽  
Maxim Levin ◽  
Mordehay Klepfish ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Drug Targets ◽  
Fas Ligand ◽  
Specific Activity ◽  
Matrix Metalloproteases ◽  
Ductal Adenocarcinoma ◽  
Enzyme Active Site ◽  
Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

scholarly journals Toward the Identification of Potential α-Ketoamide Covalent Inhibitors for SARS-CoV-2 Main Protease: Fragment-Based Drug Design and MM-PBSA Calculations

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 1004
Author(s):  
Mahmoud A. El Hassab ◽  
Mohamed Fares ◽  
Mohammed K. Abdel-Hamid Amin ◽  
Sara T. Al-Rashood ◽  
Amal Alharbi ◽  
...  
Keyword(s):  
Drug Design ◽  
Active Site ◽  
Binding Free Energy ◽  
Stable Complex ◽  
Active Site Residues ◽  
Structure Based Drug Design ◽  
Enzyme Active Site ◽  
Potential Inhibitor ◽  
Main Protease ◽  
Covalent Docking

Since December 2019, the world has been facing the outbreak of the SARS-CoV-2 pandemic that has infected more than 149 million and killed 3.1 million people by 27 April 2021, according to WHO statistics. Safety measures and precautions taken by many countries seem insufficient, especially with no specific approved drugs against the virus. This has created an urgent need to fast track the development of new medication against the virus in order to alleviate the problem and meet public expectations. The SARS-CoV-2 3CL main protease (Mpro) is one of the most attractive targets in the virus life cycle, which is responsible for the processing of the viral polyprotein and is a key for the ribosomal translation of the SARS-CoV-2 genome. In this work, we targeted this enzyme through a structure-based drug design (SBDD) protocol, which aimed at the design of a new potential inhibitor for Mpro. The protocol involves three major steps: fragment-based drug design (FBDD), covalent docking and molecular dynamics (MD) simulation with the calculation of the designed molecule binding free energy at a high level of theory. The FBDD step identified five molecular fragments, which were linked via a suitable carbon linker, to construct our designed compound RMH148. The mode of binding and initial interactions between RMH148 and the enzyme active site was established in the second step of our protocol via covalent docking. The final step involved the use of MD simulations to test for the stability of the docked RMH148 into the Mpro active site and included precise calculations for potential interactions with active site residues and binding free energies. The results introduced RMH148 as a potential inhibitor for the SARS-CoV-2 Mpro enzyme, which was able to achieve various interactions with the enzyme and forms a highly stable complex at the active site even better than the co-crystalized reference.

scholarly journals Kinetics of Microcystin-LR Removal in a Real Lake Water by UV/H2O2 Treatment and Analysis of Specific Energy Consumption

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 810
Author(s):  
Sabrina Sorlini ◽  
Carlo Collivignarelli ◽  
Marco Carnevale Miino ◽  
Francesca Maria Caccamo ◽  
Maria Cristina Collivignarelli
Keyword(s):  
Energy Consumption ◽  
Specific Energy ◽  
Lake Water ◽  
Harmful Effect ◽  
Specific Energy Consumption ◽  
World Health ◽  
Oh Radicals ◽  
H2o2 Treatment ◽  
Initial Concentration ◽  
Kinetics Of

The hepatotoxin microcystin-LR (MC-LR) represents one of the most toxic cyanotoxins for human health. Considering its harmful effect, the World Health Organization recommended a limit in drinking water (DW) of 1 µg L−1. Due to the ineffectiveness of conventional treatments present in DW treatment plants against MC-LR, advanced oxidation processes (AOPs) are gaining interest due to the high redox potential of the OH• radicals. In this work UV/H2O2 was applied to a real lake water to remove MC-LR. The kinetics of the UV/H2O2 were compared with those of UV and H2O2 showing the following result: UV/H2O2 > UV > H2O2. Within the range of H2O2 tested (0–0.9 mM), the results showed that H2O2 concentration and the removal kinetics followed an increasing quadratic relation. By increasing the initial concentration of H2O2, the consumption of oxidant also increased but, in terms of MC-LR degraded for H2O2 dosed, the removal efficiency decreased. As the initial MC-LR initial concentration increased, the removal kinetics increased up to a limit concentration (80 µg L−1) in which the presence of high amounts of the toxin slowed down the process. Operating with UV fluence lower than 950 mJ cm−2, UV alone minimized the specific energy consumption required. UV/H2O2 (0.3 mM) and UV/H2O2 (0.9 mM) were the most advantageous combination when operating with UV fluence of 950–1400 mJ cm−2 and higher than 1400 mJ cm−2, respectively.

scholarly journals Laboratory study on OH-initiated degradation kinetics of dehydroabietic acid

2015 ◽  
Vol 17 (16) ◽  
pp. 10953-10962 ◽  
Author(s):  
Chengyue Lai ◽  
Yongchun Liu ◽  
Jinzhu Ma ◽  
Qingxin Ma ◽  
Hong He
Keyword(s):  
Laboratory Study ◽  
Environmental Conditions ◽  
Degradation Kinetics ◽  
Dehydroabietic Acid ◽  
Oh Radicals ◽  
Kinetics Of

The degradation kinetics of dehydroabietic acid by OH radicals were investigated under various environmental conditions.

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