scholarly journals 1-(3-Tert-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM/MM Studies

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1608
Author(s):  
Irina V. Zueva ◽  
Sofya V. Lushchekina ◽  
Ian R. Pottie ◽  
Sultan Darvesh ◽  
Patrick Masson
Keyword(s):  
Transition State ◽  
Active Site ◽  
Enzyme Inhibitor ◽  
Organophosphorus Compounds ◽  
Palliative Therapy ◽  
Kinetic Studies ◽  
Md Simulations ◽  
Catalytic Active Site ◽  
Slow Binding Inhibitor ◽  
Covalent Intermediate

Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with Ki = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with Ki* = 0.53 nM. Reversible binding of TFK provides a long residence time, τ = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the “isomerization” step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called “transition state analog”, followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer’s disease and protection of central AChE against organophosphorus compounds.

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

scholarly journals Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin

1988 ◽  
Vol 256 (2) ◽  
pp. 357-361 ◽  
Author(s):  
P Hadváry ◽  
H Lengsfeld ◽  
H Wolfer
Keyword(s):  
Transition State ◽  
Acid Derivative ◽  
Pancreatic Lipase ◽  
Active Site ◽  
Boronic Acid ◽  
Selective Inhibitor ◽  
Covalent Intermediate ◽  
State Form ◽  
Covalent Inhibitor

Tetrahydrolipstatin inhibits pancreatic lipase from several species, including man, with comparable potency. The lipase is progressively inactivated through the formation of a long-lived covalent intermediate, probably with a 1:1 stoichiometry. The lipase substrate triolein and also a boronic acid derivative, which is presumed to be a transition-state-form inhibitor, retard the rate of inactivation. Therefore, in all probability, tetrahydrolipstatin reacts with pancreatic lipase at, or near, the substrate binding or active site. Tetrahydrolipstatin is a selective inhibitor of lipase; other hydrolases tested were at least a thousand times less potently inhibited.

scholarly journals Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site

2021 ◽  
Author(s):  
Léa El Khoury ◽  
David Mobley ◽  
Dongmei Ye ◽  
Susan Rempe
Keyword(s):  
Active Site ◽  
Binding Free Energy ◽  
Hot Spot ◽  
Circulatory System ◽  
Kinetic Studies ◽  
Md Simulations ◽  
Binding Mode ◽  
Hydrolytic Activity ◽  
Free Energy Calculations ◽  
Organophosphorus Hydrolase

<p>Organophosphorus (OP) compounds are among the most toxic of chemical substances and widely used as insecticides, pesticides, and chemical warfare agents. The most important enzyme inhibited by OP compounds is acetylcholinesterase (AChe). Inactivation of AChe function results in the accumulation of neurotransmitter, leading to death due to serious respiratory disorders. Organophosphorus hydrolase (OPH), also called phosphotriesterase, is a homo-dimeric metalloenzyme that can hydrolyze various OP agents in the circulatory system, resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is the insecticide diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon in the OPH active site. We identified a potential binding mode of paraoxon that does not match the binding mode of diethyl 4-methylbenzylphosphonate. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified 3 hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. To further assess these predictions, we then experimentally assayed single and double mutants involving these residues (D253E, H254S, I255S, D253E-H254R and D253E-I255G) for hydrolytic activity against paraoxon. Computational structural analysis of protein-substrate dynamics shows different hydrogen bonding profiles for mutants involving D253 (D253E, D253E-H254R, and D253E-I255G) compared to WT OPH. Additionally, the binding free energy calculations and the experimental kinetics (particularly, <i>k</i><sub>cat</sub> and <i>K<sub>M</sub></i>) of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced activity over WT OPH. Interestingly, our experimental results show that the activity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.</p>

Insights into transition state stabilization of the β-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants

10.1038/1852 ◽  
1998 ◽  
Vol 5 (9) ◽  
pp. 812-818 ◽  
Author(s):  
Valerie Notenboom ◽  
Camelia Birsan ◽  
Mark Nitz ◽  
David R. Rose ◽  
R. Antony J. Warren ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Transition State Stabilization ◽  
Covalent Intermediate ◽  
Active Site Mutants

scholarly journals Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site

2021 ◽  
Author(s):  
Léa El Khoury ◽  
David Mobley ◽  
Dongmei Ye ◽  
Susan Rempe
Keyword(s):  
Active Site ◽  
Binding Free Energy ◽  
Hot Spot ◽  
Circulatory System ◽  
Kinetic Studies ◽  
Md Simulations ◽  
Binding Mode ◽  
Hydrolytic Activity ◽  
Free Energy Calculations ◽  
Organophosphorus Hydrolase

<p>Organophosphorus (OP) compounds are among the most toxic of chemical substances and widely used as insecticides, pesticides, and chemical warfare agents. The most important enzyme inhibited by OP compounds is acetylcholinesterase (AChe). Inactivation of AChe function results in the accumulation of neurotransmitter, leading to death due to serious respiratory disorders. Organophosphorus hydrolase (OPH), also called phosphotriesterase, is a homo-dimeric metalloenzyme that can hydrolyze various OP agents in the circulatory system, resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is the insecticide diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon in the OPH active site. We identified a potential binding mode of paraoxon that does not match the binding mode of diethyl 4-methylbenzylphosphonate. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified 3 hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. To further assess these predictions, we then experimentally assayed single and double mutants involving these residues (D253E, H254S, I255S, D253E-H254R and D253E-I255G) for hydrolytic activity against paraoxon. Computational structural analysis of protein-substrate dynamics shows different hydrogen bonding profiles for mutants involving D253 (D253E, D253E-H254R, and D253E-I255G) compared to WT OPH. Additionally, the binding free energy calculations and the experimental kinetics (particularly, <i>k</i><sub>cat</sub> and <i>K<sub>M</sub></i>) of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced activity over WT OPH. Interestingly, our experimental results show that the activity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.</p>

scholarly journals Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site

2021 ◽  
Vol 22 (23) ◽  
pp. 12624
Author(s):  
Léa El Khoury ◽  
David L. Mobley ◽  
Dongmei Ye ◽  
Susan B. Rempe
Keyword(s):  
Binding Affinity ◽  
Active Site ◽  
Binding Free Energy ◽  
Hot Spot ◽  
Substrate Binding ◽  
Kinetic Studies ◽  
Md Simulations ◽  
Binding Mode ◽  
Free Energy Calculations ◽  
Organophosphorus Hydrolase

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.

Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies

2013 ◽  
Vol 454 (3) ◽  
pp. 387-399 ◽  
Author(s):  
Patrick Masson ◽  
Sofya Lushchekina ◽  
Lawrence M. Schopfer ◽  
Oksana Lockridge
Keyword(s):  
Osmotic Stress ◽  
Osmotic Pressure ◽  
Active Site ◽  
Md Simulations ◽  
Catalytic Triad ◽  
Wild Type ◽  
Irreversible Inhibitor ◽  
Enzyme Active Site ◽  
Diffusion Controlled ◽  
Peripheral Site

CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E′. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon≈108 M−1·min−1). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp70 in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium E⇌E′, indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E′. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His438, leading to the less reactive form E′.

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