Thermal Investigation of P-Phenylene-Bis and Phenyl Dithiocarbamate Binding to Mushroom Tyrosinase

2011 ◽  
Vol 110-116 ◽  
pp. 1970-1974
Author(s):  
Gholamreza Rezaei Behbehani ◽  
M. Mehreshtiagh
Keyword(s):  
Active Site ◽  
Phosphate Buffer ◽  
Electrostatic Interactions ◽  
Structural Changes ◽  
Copper Ions ◽  
Mushroom Tyrosinase ◽  
Binding Properties ◽  
Enzyme Active Site ◽  
Head Groups ◽  
Tyrosinase Enzyme

The binding properties and structural changes of mushroom tyrosinase enzyme, MT, due to its interaction with phenyl dithiocarbamate (I) and p-phenylene-bis dithiocarbamate (II) were investigated at 27 and 37°C in phosphate buffer (10 mmol.L-1) at pH=6.8 by isothermal titration calorimetric (ITC). Thermodynamic analysis indicated that predominant mode of interaction was hydrophobic in binding of I to MT, meanwhile the binding of II to MT essentially depends on electrostatic interactions. It seems thatII is a more potent MT inhibitor due to its two charged head groups able to chelate copper ions in the enzyme active site. It was concluded that MT has two distinct sites for p-phenylene-bis and phenyl dithiocarbamate.

scholarly journals Evaluation of p-phenylene-bis and phenyl dithiocarbamate sodium salts as inhibitors of mushroom tyrosinase.

2010 ◽  
Vol 57 (3) ◽  
Author(s):  
Ehsan Amin ◽  
Ali Akbar Saboury ◽  
Hassan Mansouri-Torshizi ◽  
Samane Zolghadri ◽  
Abdol-Khalegh Bordbar
Keyword(s):  
Sodium Salt ◽  
Tertiary Structure ◽  
Hydrophobic Interactions ◽  
Copper Ions ◽  
Water Soluble ◽  
Spectroscopic Techniques ◽  
Mushroom Tyrosinase ◽  
Enzyme Active Site ◽  
Fluorescence Studies ◽  
Head Groups

Two structurally related compounds, phenyl dithiocarbamate sodium salt (I) and p-phenylene-bis (dithiocarbamate) sodium salt (II) were prepared by reaction of the parent aniline and p-phenylenediamine with CS₂ in the presence of sodium hydroxide. These water soluble compounds were characterized by spectroscopic techniques, IR, ¹H NMR and elemental analysis. The inhibitory effects of both compounds on both activities of mushroom tyrosinase (MT) from Agricus bisporus were studied at two temperatures, 27°C and 37°C. L-3, 4-dihydroxyphenylalanine (L-DOPA), and l-tyrosine were used as natural substrates for the catecholase and cresolase enzyme reactions, respectively. Kinetic analysis confirmed noncompetitive inhibition mode of I and mixed type of II on both activities of MT; I and II inhibit MT with inhibition constants (K(I)) of 300 µM and 4 µM, respectively. Analysis of thermodynamic parameters indicated predominant involvement of hydrophobic interactions in binding of I and electrostatic ones in binding of II to MT. It seems that II is a more potent MT inhibitor due to its two charged head groups able to chelate copper ions in the enzyme active site. Intrinsic fluorescence studies as a function of concentrations of both compounds showed unexpectedly quenching of emission intensity without any shift of emission maximum. Extrinsic ANS-fluorescence indicated that only binding of I induces limited changes in the tertiary structure of MT, in agreement with the postulated hydrophobic nature of the binding mechanism.

scholarly journals A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase

2020 ◽  
Author(s):  
Yufan Wu ◽  
Stephen Fried ◽  
Steven Boxer
Keyword(s):  
Electric Field ◽  
Electric Fields ◽  
Ground State ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Stark Effect ◽  
Structural Changes ◽  
Enzymatic Catalysis ◽  
Ketosteroid Isomerase ◽  
Computational Enzyme Design

<div><p>Electrostatic interactions play a pivotal role in enzymatic catalysis and are increasingly modeled explicitly in computational enzyme design; nevertheless, they are challenging to measure experimentally. Using vibrational Stark effect (VSE) spectroscopy, we have measured electric fields inside the active site of the enzyme ketosteroid isomerase (KSI). These studies have shown that these fields can be unusually large, but it has been unclear to what extent they specifically stabilize the transition state (TS) relative to a ground state (GS). In the following, we use crystallography and computational modeling to show that KSI’s intrinsic electric field is nearly perfectly oriented to stabilize the geometry of its reaction’s TS. Moreover, we find that this electric field adjusts the orientation of its substrate in the ground state so that the substrate needs to only undergo minimal structural changes upon activation to its TS. This work provides evidence that the active site electric field in KSI is preorganized to facilitate catalysis and provides a template for how electrostatic preorganization can be measured in enzymatic systems. <br></p></div>

scholarly journals Dual effects of presynaptic membrane mimetics on α-synuclein amyloid aggregation

2021 ◽  
Author(s):  
Yuxi Lin ◽  
Dai Ito ◽  
Je Min Yoo ◽  
Mi Hee Lim ◽  
Woo Kyung Yu ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Structural Changes ◽  
Dependent Manner ◽  
Binding Properties ◽  
Intrinsically Disordered ◽  
Membrane Surfaces ◽  
Amyloid Fibrillation ◽  
Presynaptic Vesicle ◽  
Helical Folding ◽  
Dose Dependent

Aggregation of intrinsically disordered α-synuclein (αSN) under various conditions is closely related to synucleinopathies. Although various biological membranes have shown to alter the structure and aggregation propensity of αSN, a thorough understanding of the molecular and mechanical mechanism of amyloidogenesis in membranes remains unanswered. Herein, we examined the structural changes, binding properties, and amyloidogenicity of three variations of αSN mutants under two types of liposomes, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and presynaptic vesicle mimetic (Mimic) membranes. While neutrally charged DOPC membranes elicited marginal changes in the structure and amyloid fibrillation of αSNs, negatively charged Mimic membranes induced dramatic helical folding and biphasic amyloid generation. At low concentration of Mimic membranes, the amyloid fibrillation of αSNs was promoted in a dose-dependent manner. However, further increases in the concentration constrained the fibrillation process. These results suggest the dual effect of Mimic membranes on regulating the amyloidogenesis of αSN, which is rationalized by the amyloidogenic structure of αSN and condensation-dilution of local αSN concentration. Finally, we propose physicochemical properties of αSN and membrane surfaces, and their propensity to drive electrostatic interactions as decisive factors of amyloidogenesis.

scholarly journals A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase

2020 ◽  
Author(s):  
Yufan Wu ◽  
Stephen Fried ◽  
Steven Boxer
Keyword(s):  
Electric Field ◽  
Electric Fields ◽  
Ground State ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Stark Effect ◽  
Structural Changes ◽  
Enzymatic Catalysis ◽  
Ketosteroid Isomerase ◽  
Computational Enzyme Design

<div><p>Electrostatic interactions play a pivotal role in enzymatic catalysis and are increasingly modeled explicitly in computational enzyme design; nevertheless, they are challenging to measure experimentally. Using vibrational Stark effect (VSE) spectroscopy, we have measured electric fields inside the active site of the enzyme ketosteroid isomerase (KSI). These studies have shown that these fields can be unusually large, but it has been unclear to what extent they specifically stabilize the transition state (TS) relative to a ground state (GS). In the following, we use crystallography and computational modeling to show that KSI’s intrinsic electric field is nearly perfectly oriented to stabilize the geometry of its reaction’s TS. Moreover, we find that this electric field adjusts the orientation of its substrate in the ground state so that the substrate needs to only undergo minimal structural changes upon activation to its TS. This work provides evidence that the active site electric field in KSI is preorganized to facilitate catalysis and provides a template for how electrostatic preorganization can be measured in enzymatic systems. <br></p></div>

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.

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