37 results on '"Jiayun Pang"'
Search Results
2. Delivering Antisense Oligonucleotides across the Blood‐Brain Barrier by Tumor Cell‐Derived Small Apoptotic Bodies
- Author
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Yulian Wang, Jiayun Pang, Qingyun Wang, Luocheng Yan, Lintao Wang, Zhen Xing, Chunming Wang, Junfeng Zhang, and Lei Dong
- Subjects
blood‐brain barrier ,CD44v6 ,small apoptotic bodies ,transcytosis ,tumor cells ,Science - Abstract
Abstract The blood‐brain barrier (BBB) is the most restrictive and complicated barrier that keeps most biomolecules and drugs from the brain. An efficient brain delivery strategy is urgently needed for the treatment of brain diseases. Based on the studies of brain‐targeting extracellular vesicles (EVs), the potential of using small apoptotic bodies (sABs) from brain metastatic cancer cells for brain‐targeting drug delivery is explored. It is found that anti‐TNF‐α antisense oligonucleotide (ASO) combined with cationic konjac glucomannan (cKGM) can be successfully loaded into sABs via a transfection/apoptosis induction process and that the sABs generated by B16F10 cells have an extraordinarily high brain delivery efficiency. Further studies suggest that ASO‐loaded sABs (sCABs) are transcytosed by b. End3 (brain microvascular endothelial cells, BMECs) to penetrate the BBB, which is mediated by CD44v6, and eventually taken up by microglial cells in the brain. In a Parkinson's disease (PD) mouse model, sCABs dramatically ameliorate PD symptoms via the anti‐inflammatory effect of ASO. This study suggests that sABs from brain metastatic cancer cells are excellent carriers for brain‐targeted delivery, as they have not only an extraordinary delivery efficiency but also a much higher scale‐up production potential than other EVs.
- Published
- 2021
- Full Text
- View/download PDF
3. Specialising and Analysing Instruction-Tuned and Byte-Level Language Models for Organic Reaction Prediction.
- Author
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Jiayun Pang and Ivan Vulic
- Published
- 2024
- Full Text
- View/download PDF
4. Drug Delivery: Delivering Antisense Oligonucleotides across the Blood‐Brain Barrier by Tumor Cell‐Derived Small Apoptotic Bodies (Adv. Sci. 13/2021)
- Author
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Luocheng Yan, Zhen Xing, Chunming Wang, Lintao Wang, Lei Dong, Qingyun Wang, Jiayun Pang, Yulian Wang, and Junfeng Zhang
- Subjects
business.industry ,General Chemical Engineering ,Inside Back Cover ,General Engineering ,General Physics and Astronomy ,Medicine (miscellaneous) ,Tumor cells ,Blood–brain barrier ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Text mining ,medicine.anatomical_structure ,Apoptosis ,Antisense oligonucleotides ,Drug delivery ,Cancer research ,Medicine ,General Materials Science ,business - Abstract
Brain, guarded by the most rigorous barrier — blood‐brain barrier (BBB), is the most critical and sophisticated control center in the body. The dead bodies from melanoma cells, small apoptotic bodies with membrane embedded CD44v6 molecules could pass through BBB and deliver drugs for the cure of Parkinson's disease. The apoptotic body might be a new type of carrier for efficient in vivo drug delivery. More details can be found in article number 2004929 by Chunming Wang, Junfeng Zhang, Lei Dong, and co‐workers. [Image: see text]
- Published
- 2021
5. Delivering Antisense Oligonucleotides across the Blood‐Brain Barrier by Tumor Cell‐Derived Small Apoptotic Bodies
- Author
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Lintao Wang, Yulian Wang, Junfeng Zhang, Zhen Xing, Chunming Wang, Jiayun Pang, Lei Dong, Qingyun Wang, and Luocheng Yan
- Subjects
Male ,blood‐brain barrier ,General Chemical Engineering ,Science ,tumor cells ,General Physics and Astronomy ,Medicine (miscellaneous) ,transcytosis ,Tumor cells ,Blood–brain barrier ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Mannans ,Extracellular Vesicles ,Mice ,medicine ,Animals ,General Materials Science ,Research Articles ,Brain Neoplasms ,Chemistry ,General Engineering ,CD44v6 ,Transfection ,Oligonucleotides, Antisense ,Thionucleotides ,Mice, Inbred C57BL ,Disease Models, Animal ,medicine.anatomical_structure ,Transcytosis ,Blood-Brain Barrier ,Apoptosis ,Drug delivery ,Antisense oligonucleotides ,Cancer cell ,Cancer research ,small apoptotic bodies ,Research Article - Abstract
The blood‐brain barrier (BBB) is the most restrictive and complicated barrier that keeps most biomolecules and drugs from the brain. An efficient brain delivery strategy is urgently needed for the treatment of brain diseases. Based on the studies of brain‐targeting extracellular vesicles (EVs), the potential of using small apoptotic bodies (sABs) from brain metastatic cancer cells for brain‐targeting drug delivery is explored. It is found that anti‐TNF‐α antisense oligonucleotide (ASO) combined with cationic konjac glucomannan (cKGM) can be successfully loaded into sABs via a transfection/apoptosis induction process and that the sABs generated by B16F10 cells have an extraordinarily high brain delivery efficiency. Further studies suggest that ASO‐loaded sABs (sCABs) are transcytosed by b. End3 (brain microvascular endothelial cells, BMECs) to penetrate the BBB, which is mediated by CD44v6, and eventually taken up by microglial cells in the brain. In a Parkinson's disease (PD) mouse model, sCABs dramatically ameliorate PD symptoms via the anti‐inflammatory effect of ASO. This study suggests that sABs from brain metastatic cancer cells are excellent carriers for brain‐targeted delivery, as they have not only an extraordinary delivery efficiency but also a much higher scale‐up production potential than other EVs., Cancer cells that spread to the brain naturally produce nano‐sized “apoptotic bodies” (ABs), which can package nucleic acid drugs efficiently (>21.3%), cross the brain‐blood barrier via transcytosis mediated by CD44v6 exposed on ABs, and deliver anti‐inflammatory nucleotides into microglial cells. These ABs show potential as carriers for the treatment and diagnosis of brain diseases.
- Published
- 2021
6. Development of boronic acid-functionalized mesoporous silica-coated core/shell magnetic microspheres with large pores for endotoxin removal
- Author
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Peixuan Zhao, Jiayun Pang, Yibing Ji, Wenxian Fu, and Wei Li
- Subjects
Chemical structure ,Buffers ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Ferric Compounds ,Analytical Chemistry ,chemistry.chemical_compound ,Magnetics ,Adsorption ,X-Ray Diffraction ,Desorption ,Chromatography ,Chemistry ,Elution ,010401 analytical chemistry ,Organic Chemistry ,General Medicine ,Mesoporous silica ,Reference Standards ,Silicon Dioxide ,Boronic Acids ,Microspheres ,0104 chemical sciences ,Endotoxins ,Transmission electron microscopy ,Mesoporous material ,Porosity ,Boronic acid ,Nuclear chemistry - Abstract
Endotoxins are found almost everywhere and possess high toxicity in vivo and in vitro. Here we design a novel boronate affinity material, called boronic acid-functionalized mesoporous silica-coated core/shell magnetic microspheres (Fe3O4@nSiO2@mSiO2-BA) with large pores (pore size > 20 nm) based on the chemical structure and physical properties of endotoxins, for facile and highly efficient removal of endotoxins. Dual modes for endotoxin removal were proposed and confirmed in this work: the endotoxin aggregates with size 20 nm) were absorbed on the outer surface of the prepared material based on boronate affinity. Transmission electron microscopy (TEM), X-ray diffraction (XRD), nitrogen adsorption/desorption isotherms and Fourier transform infrared (FT-IR) spectroscopy confirm that Fe3O4@nSiO2@mSiO2-BA microspheres possess core/shell structure, uniform diameter (520 nm), high surface area (205.57 m2/g), large mesopores (21.8 nm) and boronic acid ligands. The purification procedures of Fe3O4@nSiO2@mSiO2-BA microspheres for endotoxin were optimized, and 50 mM NH4HCO3 (pH 8.0) and 0.05 M fructose were selected as loading/washing, elution buffers, respectively. The binding capacity of Fe3O4@nSiO2@mSiO2-BA microspheres for endotoxin was calculated to be 60.84 EU/g under the optimized conditions. Finally, the established analytical method was applied to remove endotoxins from plasmid DNA. After endotoxin removal, the endotoxin content in plasmid DNA was reduced from 0.0026 to 0.0006 EU/mL for two-fold concentration, and from 0.0088 to 0.0022 EU/mL for five-fold concentration after binding, respectively. Additional advantages of the prepared boronate affinity material include excellent stability, reusability/repeatability, and low cost. Boronate affinity materials with large pores could thus prove to be powerful adsorbents for endotoxin removal and the potential applications in the aspects of biological research, pharmaceutical industry, and life health.
- Published
- 2019
7. New insights into the molecular mechanism of methanol-induced inactivation ofThermomyces lanuginosuslipase: a molecular dynamics simulation study
- Author
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Peter Kamp Busk, Xiaoxue Tong, Lene Lange, and Jiayun Pang
- Subjects
0301 basic medicine ,General Chemical Engineering ,03 medical and health sciences ,Molecular dynamics ,symbols.namesake ,chemistry.chemical_compound ,Organic chemistry ,QD ,General Materials Science ,Lipase ,QA ,biology ,Hydrogen bond ,Active site ,General Chemistry ,Condensed Matter Physics ,Solvent ,030104 developmental biology ,Catalytic cycle ,chemistry ,Modeling and Simulation ,biology.protein ,symbols ,Methanol ,van der Waals force ,Information Systems - Abstract
Methanol intolerance of lipase is a major limitation in lipase-catalyzed methanolysis reactions. In this study, to understand the molecular mechanism of methanol-induced inactivation of lipases, we performed molecular dynamics (MD) simulations of Thermomyces lanuginosus lipase (TLL) in water and methanol and compared the observed structural and dynamic properties. The solvent accessibility analysis showed that in methanol, polar residues tended to be buried away from the solvent while non-polar residues tended to be more solvent-exposed in comparison to those in water. Moreover, we observed that in methanol, the van der Waals packing of the core residues in two hydrophobic regions of TLL became weak. Additionally, the catalytically relevant hydrogen bond between Asp201 OD2 and His258 ND1 in the active site was broken when the enzyme was solvated in methanol. This may affect the stability of the tetrahedral intermediates in the catalytic cycle of TLL. Furthermore, compared to those in water, some enzyme surface residues displayed enhanced movement in methanol with higher Cα root-mean-square atomic positional fluctuation values. One of such methanol-affecting surface residues (Ile241) was chosen for mutation, and MD simulation of the I241E mutant in methanol was conducted. The structural analysis of the mutant showed that replacing a non-polar surface residue with an acidic one at position 241 contributed to the stabilization of enzyme structure in methanol. Ultimately, these results, while providing molecular-level insights into the destabilizing effect of methanol on TLL, highlight the importance of surface residue redesign to improve the stability of lipases in methanol environments.
- Published
- 2015
8. Drug–polymer intermolecular interactions in hot-melt extruded solid dispersions
- Author
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Mohammed Maniruzzaman, Dennis Douroumis, David J. Morgan, Jiayun Pang, Martin J. Snowden, and Andrew P. Mendham
- Subjects
Anions ,Models, Molecular ,Hot Temperature ,Magnetic Resonance Spectroscopy ,Surface Properties ,Drug Compounding ,Acrylic Resins ,Pharmaceutical Science ,Miscibility ,chemistry.chemical_compound ,Polymethacrylic Acids ,X-Ray Diffraction ,Cations ,Polymer chemistry ,Copolymer ,Transition Temperature ,Methyl methacrylate ,chemistry.chemical_classification ,Drug Carriers ,Calorimetry, Differential Scanning ,Chemistry ,Photoelectron Spectroscopy ,Intermolecular force ,Cationic polymerization ,Hydrogen Bonding ,Polymer ,Propranolol ,Diphenhydramine ,Solubility ,Methacrylic acid ,Microscopy, Electron, Scanning ,Ethyl acrylate - Abstract
The purpose of the study was to investigate and identify the interactions within solid dispersions of cationic drugs and anionic polymers processed by hot-melt extrusion (HME) technique. Propranolol HCl (PRP) and diphenhydramine HCl (DPD) were used as model cationic active substances while pH sensitive anionic methacrylic acid based methyl methacrylate copolymers Eudragit L100® (L100) and ethyl acrylate copolymer Eudragit L100-55 (Acryl EZE) (L100-55) were used as polymeric carriers. The extrudates were further characterised using various physicochemical characterisation techniques to determine the morphology, the drug state within the polymer matrices and the type of drug–polymer interactions. Molecular modelling predicted the existence of two possible H-bonding types while the X-ray photon spectroscopy (XPS) advanced surface analysis of the extrudates revealed intermolecular ionic interactions between the API amino functional groups and the polymer carboxylic groups through the formation of hydrogen bonding. The magnitude of the intermolecular interactions varied according to the drug–polymer miscibility.
- Published
- 2013
9. Assignment of the vibrational spectra of enzyme-bound tryptophan tryptophyl quinones using a combined QM/MM approach
- Author
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Jiayun Pang, Scrutton, Nigel S., de Visser, Sam P., and Sutcliffe, Michael J.
- Subjects
Aromatic amines -- Chemical properties ,Fourier transform infrared spectroscopy -- Usage ,Quantum theory -- Usage ,Quinone -- Chemical properties ,Quinone -- Structure ,Tryptophan -- Structure ,Tryptophan -- Chemical properties ,Vibrational spectra -- Analysis ,Chemicals, plastics and rubber industries - Published
- 2010
10. Deep tunneling dominates the biologically important hydride transfer reaction from NADH to FMN in morphinone reductase
- Author
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Jiayun Pang, Sam Hay, Scrutton, Nigel S., and Sutcliffe, Michael J.
- Subjects
Flavin mononucleotide -- Chemical properties ,NAD (Coenzyme) -- Chemical properties ,Oxidoreductases -- Chemical properties ,Transition state (Chemistry) -- Analysis ,Chemistry - Abstract
The variational transition-state theory calculations are employed to study the hydride transfer taking place from nicotinamide adenine dinucleotide (NADH) to flavin mononucleotide (FMN) in morphinone reductase. The transfer is shown to be biologically important and is completely dominated by the deep tunneling phenomenon.
- Published
- 2008
11. Temperature-dependent study reveals that dynamics of hydrophobic residues plays an important functional role in the mitochondrial Tim9-Tim10 complex
- Author
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Ekaterina Ivanova, Michael J. Sutcliffe, Hui Lu, Jiayun Pang, Jim Warwicker, Guanhua Yan, and Thomas A. Jowitt
- Subjects
Conformational change ,biology ,Chemistry ,Mitochondrial intermembrane space ,Tim9-Tim10 complex ,Biochemistry ,Protein–protein interaction ,Hydrophobic effect ,Crystallography ,Molecular dynamics ,Structural Biology ,Biophysics ,biology.protein ,Translocase ,Inner mitochondrial membrane ,Molecular Biology - Abstract
Protein-protein interaction is a fundamental process in all major biological processes. The hexameric Tim9-Tim10 (translocase of inner membrane) complex of the mitochondrial intermembrane space plays an essential chaperone-like role during import of mitochondrial membrane proteins. However, little is known about the functional mechanism of the complex because the interaction is weak and transient. This study investigates how electrostatic and hydrophobic interactions affect the conformation and function of the complex at physiological temperatures, using both experimental and computational methods. The results suggest that, first, different complex conformational states exist at equilibrium, and the major difference between these states is the degree of hydrophobic interactions. Second, the conformational change mimics the biological activity of the complex as measured by substrate binding at the same temperatures. Finally, molecular dynamics simulation and detailed energy decomposition analysis provided supporting evidence at the atomic level for the presence of an excited state of the complex, the formation of which is largely driven by the disruption of hydrophobic interactions. Taken together, this study indicates that the dynamics of the hydrophobic residues plays an important role in regulating the function of the Tim9-Tim10 complex. Proteins 2011. © 2011 Wiley Periodicals, Inc.
- Published
- 2011
12. Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines
- Author
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Jiayun Pang, Stephen E. J. Rigby, Dominic P. H. M. Heuts, Nigel S. Scrutton, and Jennet Gummadova
- Subjects
Amine oxidase ,Quantitative structure–activity relationship ,Chemistry ,Stereochemistry ,Substrate (chemistry) ,Cell Biology ,Biochemistry ,Turnover number ,Enzyme catalysis ,chemistry.chemical_compound ,Benzylamine ,Kinetic isotope effect ,Enzyme kinetics ,Molecular Biology - Abstract
Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pKa values that defined a bell-shaped plot of turnover number kcat,app as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (kcat/Km)app on pH revealed a single pKa value (∼9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (kcat/Km)app over the pH range of 6 to 10 was observed with d2-benzylamine. Over the same pH range, the KIE on kcat was found to be close to unity. The unusual KIE values on (kcat/Km)app were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on kcat,app (8.01 ± 0.28 with phenylethylamine), indicating that C–H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism.
- Published
- 2011
13. Mutagenesis of morphinone reductase induces multiple reactive configurations and identifies potential ambiguity in kinetic analysis of enzyme tunneling mechanisms
- Author
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Pudney, Christopher R., Hay, Sam, Jiayun Pang, Costello, Claire, Leys, David, Sutcliffe, Michael J., and Scrutton, Nigel S.
- Subjects
Morphine -- Structure ,Morphine -- Spectra ,Charge transfer -- Research ,NAD (Coenzyme) -- Structure ,NAD (Coenzyme) -- Chemical properties ,Chemistry - Abstract
Stopped-flow spectroscopy has demonstrated that reduction of the FMN cofactor by NADH in N189A morphinone reductase (MR) in multiphasic, thus identifying four different reactive configurations of the MR-NADH complex. The analysis has helped in deriving the mechanistic information regarding multiple reactive configurations of the same enzyme-coenzyme complex by using stopped-flow methods.
- Published
- 2007
14. Deep Tunneling Dominates the Biologically Important Hydride Transfer Reaction from NADH to FMN in Morphinone Reductase
- Author
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Nigel S. Scrutton, Jiayun Pang, Michael J. Sutcliffe, and Sam Hay
- Subjects
Morphinone reductase ,Flavin Mononucleotide ,Chemistry ,Hydride ,General Chemistry ,Nicotinamide adenine dinucleotide ,NAD ,Photochemistry ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Transition state theory ,Colloid and Surface Chemistry ,Reaction rate constant ,Bacterial Proteins ,Deuterium ,Kinetic isotope effect ,Quantum Theory ,Methylamine dehydrogenase ,Oxidoreductases - Abstract
The temperature dependence of the primary kinetic isotope effect (KIE), combined temperature-pressure studies of the primary KIE, and studies of the alpha-secondary KIE previously led us to infer that hydride transfer from nicotinamide adenine dinucleotide to flavin mononucleotide in morphinone reductase proceeds via environmentally coupled hydride tunneling. We present here a computational analysis of this hydride transfer reaction using QM/MM molecular dynamics simulations and variational transition-state theory calculations. Our calculated primary and secondary KIEs are in good agreement with the corresponding experimental values. Although the experimentally observed KIE lies below the semiclassical limit, our calculations suggest that approximately 99% of the reaction proceeds via tunneling: this is the first "deep tunneling" reaction observed for hydride transfer. We also show that the dominant tunneling mechanism is controlled by the isotope at the primary rather than the secondary position: with protium in the primary position, large-curvature tunneling dominates, whereas with deuterium in this position, small-curvature tunneling dominates. Also, our study is consistent with tunneling being preceded by reorganization: in the reactant, the rings of the nicotinamide and isoalloxazine moieties are stacked roughly parallel to each other, and as the system moves toward a "tunneling-ready" configuration, the nicotinamide ring rotates to become almost perpendicular to the isoalloxazine ring.
- Published
- 2008
15. Atomistic insight into the origin of the temperature-dependence of kinetic isotope effects and H-tunnelling in enzyme systems is revealed through combined experimental studies and biomolecular simulation
- Author
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Laura Masgrau, Jiayun Pang, Parvinder Hothi, Sam Hay, Christopher R. Pudney, Nigel S. Scrutton, Linus O. Johannissen, David Leys, and Michael J. Sutcliffe
- Subjects
Models, Molecular ,Chemistry ,Protein dynamics ,Temperature ,Kinetic energy ,Biochemistry ,Enzymes ,Enzyme catalysis ,Kinetics ,Isotopes ,Chemical physics ,Kinetic isotope effect ,Physical chemistry ,Computer Simulation ,Quantum ,Quantum tunnelling - Abstract
The physical basis of the catalytic power of enzymes remains contentious despite sustained and intensive research efforts. Knowledge of enzyme catalysis is predominantly descriptive, gained from traditional protein crystallography and solution studies. Our goal is to understand catalysis by developing a complete and quantitative picture of catalytic processes, incorporating dynamic aspects and the role of quantum tunnelling. Embracing ideas that we have spearheaded from our work on quantum mechanical tunnelling effects linked to protein dynamics for H-transfer reactions, we review our recent progress in mapping macroscopic kinetic descriptors to an atomistic understanding of dynamics linked to biological H-tunnelling reactions.
- Published
- 2008
16. Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function
- Author
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Qi, Wang, Swee Kim, Ang, Efrain, Ceh-Pavia, Jiayun, Pang, and Hui, Lu
- Subjects
Models, Molecular ,Protein Folding ,Original Paper ,Saccharomyces cerevisiae Proteins ,Mutation, Missense ,Tryptophan ,tryptophan residue ,Saccharomyces cerevisiae ,Original Papers ,flavin-adenine dinucleotide (FAD) binding ,Catalysis ,Mitochondrial Proteins ,mitochondria ,Amino Acid Substitution ,Computer Simulation ,Oxidoreductases Acting on Sulfur Group Donors ,thiol oxidase - Abstract
Erv1 (essential for respiration and viability 1) is a FAD-dependent sulphydryl oxidase with a tryptophan-rich catalytic domain. We show that Trp95 and Trp183 are important for stabilizing the folding, FAD-binding, and function of Erv1, whilst other four tryptophan residues are not functionally important., Erv1 is an FAD-dependent thiol oxidase of the ERV (essential for respiration and viability)/ALR (augmenter of liver regeneration) sub-family and an essential component of the mitochondrial import and assembly pathway. Erv1 contains six tryptophan residues, which are all located in the highly conserved C-terminal FAD-binding domain. Though important structural roles were predicted for the invariable Trp95, no experimental study has been reported. In the present study, we investigated the structural and functional roles of individual tryptophan residues of Erv1. Six single tryptophan-to-phenylalanine yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from Escherichia coli. Their effects on folding, FAD-binding and Erv1 activity were characterized. Our results showed that Erv1W95F has the strongest effect on the stability and function of Erv1 and followed by Erv1W183F. Erv1W95F results in a decrease in the Tm of Erv1 by 23°C, a significant loss of the oxidase activity and thus causing cell growth defects at both 30°C and 37°C. Erv1W183F induces changes in the oligomerization state of Erv1, along with a pronounced effect on the stability of Erv1 and its function at 37°C, whereas the other mutants had no clear effect on the function of Erv1 including the highly conserved Trp157 mutant. Finally, computational analysis indicates that Trp95 plays a key role in stabilizing the isoalloxazine ring to interact with Cys133. Taken together, the present study provided important insights into the molecular mechanism of how thiol oxidases use FAD in catalysing disulfide bond formation.
- Published
- 2015
17. Molecular modeling as a predictive tool for the development of solid dispersions
- Author
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Dennis Douroumis, David J. Morgan, Mohammed Maniruzzaman, and Jiayun Pang
- Subjects
Models, Molecular ,Materials science ,Hot Temperature ,Molecular model ,Polymers ,Chemistry, Pharmaceutical ,Drug Compounding ,Binding energy ,Pharmaceutical Science ,Thermodynamics ,Flory–Huggins solution theory ,Miscibility ,RS ,Drug Stability ,X-Ray Diffraction ,Drug Discovery ,Organic chemistry ,Thermal analysis ,chemistry.chemical_classification ,Calorimetry, Differential Scanning ,Photoelectron Spectroscopy ,Intermolecular force ,Polymer ,Hildebrand solubility parameter ,chemistry ,Models, Chemical ,Pharmaceutical Preparations ,Solubility ,Drug Design ,Molecular Medicine ,Quantum Theory ,Crystallization ,Dimerization ,Protein Binding - Abstract
In this study molecular modelling is introduced as a novel approach for the development of pharmaceutical solid dispersions. A computational model based on quantum mechanical (QM) calculations was used to predict the miscibility of various drugs in various polymers by predicting the binding strength between the drug and dimeric form of the polymer. The drug/polymer miscibility was also estimated by using traditional approaches such as Van Krevelen/Hoftyzer and Bagley solubility parameters or Flory Huggins interaction parameter in comparison to the molecular modelling approach. The molecular modelling studies predicted successfully the drug-polymer binding energies and the preferable site of interaction between the functional groups. The drug-polymer miscibility and the physical state of bulk materials, physical mixtures and solid dispersions were determined by thermal analysis (DSC/MTDSC) and X-ray diffraction. The produced solid dispersions were analysed by X-ray photoelectron spectroscopy (XPS), which confirmed not only the exact type of the intermolecular interactions between the drug-polymer functional groups but also the binding strength by estimating the N-coefficient values. The findings demonstrate that QM-based molecular modelling is a powerful tool to predict the strength and type of intermolecular interactions in a range of drug/polymeric systems for the development of solid dispersions.
- Published
- 2015
18. Barrier Compression Enhances an Enzymatic Hydrogen‐Transfer Reaction
- Author
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Michael J. Sutcliffe, Tom A. McGrory, Christopher R. Pudney, Jiayun Pang, Sam Hay, and Nigel S. Scrutton
- Subjects
Morphinone reductase ,Ion Transport ,biology ,Hydrogen ,Flavin Mononucleotide ,Stereochemistry ,Hydrostatic pressure ,Flavin mononucleotide ,Active site ,chemistry.chemical_element ,General Chemistry ,NAD ,Photochemistry ,Catalysis ,Enzyme catalysis ,chemistry.chemical_compound ,Molecular dynamics ,Bacterial Proteins ,chemistry ,Biocatalysis ,Hydrostatic Pressure ,biology.protein ,Oxidoreductases - Abstract
Putting the squeeze on: Hydrostatic pressure causes a shortening of the charge-transfer bond in the binary complex of morphinone reductase and NADH(4) (see diagram). Molecular dynamics simulations suggest that pressure reduces the average reaction barrier width by restricting the conformational space available to the flavin mononucleotide and NADH within the active site. The apparent rate of catalysis increases with pressure.
- Published
- 2009
19. Barrier Compression Enhances an Enzymatic Hydrogen‐Transfer Reaction
- Author
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Sam Hay, Christopher R. Pudney, Tom A. McGrory, Jiayun Pang, Michael J. Sutcliffe, and Nigel S. Scrutton
- Subjects
General Medicine - Published
- 2009
20. Large-scale domain conformational change is coupled to the activation of the Co-C bond in the B12-dependent enzyme ornithine 4,5-aminomutase: a computational study
- Author
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Xin Li, Nigel S. Scrutton, Keiji Morokuma, Jiayun Pang, and Michael J. Sutcliffe
- Subjects
Steric effects ,Models, Molecular ,Conformational change ,Coenzyme B ,Stereochemistry ,Molecular Conformation ,Isomerase ,Molecular Dynamics Simulation ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,medicine ,Intramolecular Transferases ,biology ,Corrin ,Active site ,Stereoisomerism ,General Chemistry ,Cobalt ,Adenosylcobalamin ,Carbon ,Homolysis ,chemistry ,biology.protein ,Cobamides ,medicine.drug - Abstract
We present here an energetic and atomistic description of how D-ornithine 4,5-aminomutase (OAM), an adenosylcobalamin (AdoCbl; coenzyme B(12))-dependent isomerase, employs a large-scale protein domain conformational change to orchestrate the homolytic rupture of the Co-C bond. Our results suggest that in going from the open form (catalytically inactive) to the closed form (catalytically active), the Rossmann domain of OAM effectively approaches the active site as a rigid body. It undergoes a combination of a ~52° rotation and a ~14 A translation to bring AdoCbl-initially positioned ~25 A away-into the active-site cavity. This process is coupled to repositioning of the Ado moiety of AdoCbl from the eastern conformation to the northern conformation. Combined quantum mechanics and molecular mechanics calculations further indicate that in the open form, the protein environment does not impact significantly on the Co-C bond homolytic rupture, rendering it unusually stable, and thus catalytically inactive. Upon formation of the closed form, the Co-C bond is activated through the synergy of steric and electrostatic effects arising from tighter interactions with the surrounding enzyme. The more pronounced effect of the protein in the closed form gives rise to an elongated Co-C bond (by 0.03 A), puckering of the ribose and increased "strain" energy on the Ado group and to a lesser extent the corrin ring. Our computational studies reveal novel strategies employed by AdoCbl-dependent enzymes in the control of radical catalysis.
- Published
- 2012
21. Reaction of vascular adhesion protein-1 (VAP-1) with primary amines: mechanistic insights from isotope effects and quantitative structure-activity relationships
- Author
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Dominic P H M, Heuts, Jennet O, Gummadova, Jiayun, Pang, Stephen E J, Rigby, and Nigel S, Scrutton
- Subjects
Benzylamines ,Kinetics ,Structure-Activity Relationship ,HEK293 Cells ,Models, Chemical ,Enzymology ,Humans ,Amine Oxidase (Copper-Containing) ,Hydrogen-Ion Concentration ,Cell Adhesion Molecules - Abstract
Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pK(a) values that defined a bell-shaped plot of turnover number k(cat,app) as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (k(cat)/K(m))(app) on pH revealed a single pK(a) value (∼9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (k(cat)/K(m))(app) over the pH range of 6 to 10 was observed with d(2)-benzylamine. Over the same pH range, the KIE on k(cat) was found to be close to unity. The unusual KIE values on (k(cat)/K(m))(app) were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on k(cat,app) (8.01 ± 0.28 with phenylethylamine), indicating that C-H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism.
- Published
- 2011
22. New insights into the multi-step reaction pathway of the reductive half-reaction catalysed by aromatic amine dehydrogenase: a QM/MM study
- Author
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Michael J. Sutcliffe, Jiayun Pang, Sam P. de Visser, and Nigel S. Scrutton
- Subjects
Models, Molecular ,Oxidoreductases Acting on CH-NH Group Donors ,Half-reaction ,Proton ,Aromatic amine dehydrogenase ,Chemistry ,Metals and Alloys ,Energy landscape ,General Chemistry ,Reaction intermediate ,Photochemistry ,Models, Biological ,Catalysis ,Tryptamines ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,QM/MM ,Materials Chemistry ,Ceramics and Composites ,Stepwise reaction ,Quantum Theory - Abstract
Computational insight into the multi-step reaction cycle of aromatic amine dehydrogenase is presented, identifying the energy landscape and pathway for multiple proton transfers. This atomistic picture of the reaction sequence--including short-lived reaction intermediates and a stepwise reaction mechanism--bridges the gap between a small number of crystallographic snapshots.
- Published
- 2010
23. Integrating Computational Methods with Experiment Uncovers the Role of Dynamics in Enzyme-Catalysed H-Tunnelling Reactions
- Author
-
Michael J. Sutcliffe, Linus O. Johannissen, Nigel S. Scrutton, Jiayun Pang, and Sam Hay
- Subjects
Molecular dynamics ,Morphinone reductase ,Transition state theory ,Materials science ,Aromatic amine dehydrogenase ,Chemical physics ,Dynamics (mechanics) ,Quantum tunnelling - Abstract
We review the role of dynamics in enzyme catalysed H-tunnelling reactions with particular focus on the integration of computational methods with experimental and numerical modelling studies. We show that H-tunnelling requires compressive motion along the H-transfer coordinate and these reactions can be modelled successfully using vibrationally-coupled H-tunnelling models in which barrier compression is driven by fast motions within the enzyme–substrate complex.
- Published
- 2010
24. Parallel pathways and free-energy landscapes for enzymatic hydride transfer probed by hydrostatic pressure
- Author
-
Nigel S. Scrutton, Michael J. Sutcliffe, Tom A. McGrory, Jiayun Pang, Sam Hay, David Leys, Pierre Lafite, and Christopher R. Pudney
- Subjects
Stereochemistry ,Flavin Mononucleotide ,Hydrostatic pressure ,Population ,Crystallography, X-Ray ,Biochemistry ,Molecular dynamics ,Structure-Activity Relationship ,Bacterial Proteins ,Oxidoreductase ,Hydrostatic Pressure ,Computer Simulation ,education ,Molecular Biology ,chemistry.chemical_classification ,Morphinone reductase ,education.field_of_study ,Hydride ,Organic Chemistry ,Energy landscape ,Biological Transport ,NAD ,Kinetics ,Directed mutagenesis ,chemistry ,Amino Acid Substitution ,Chemical physics ,Biocatalysis ,Molecular Medicine ,Thermodynamics ,Mutant Proteins ,Oxidoreductases ,Hydrogen - Abstract
Mutation of an active-site residue in morphinone reductase leads to a conformationally rich landscape that enhances the rate of hydride transfer from NADH to FMN at standard pressure (1 bar). Increasing the pressure causes interconversion between different conformational substates in the mutant enzyme. While high pressure reduces the donor-acceptor distance in the wild-type enzyme, increased conformational freedom "dampens" its effect in the mutant.We show that hydride transfer from NADH to FMN catalysed by the N189A mutant of morphinone reductase occurs along parallel "chemical" pathways in a conformationally rich free-energy landscape. We have developed experimental kinetic and spectroscopic tools by using hydrostatic pressure to explore this free-energy landscape. The crystal structure of the N189A mutant enzyme in complex with the unreactive coenzyme analogue NADH(4) indicates that the nicotinamide moiety of the analogue is conformationally less restrained than the corresponding structure of the wild-type NADH(4) complex. This increased degree of conformational freedom in the N189A enzyme gives rise to the concept of multiple reactive configurations (MRCs), and we show that the relative population of these states across the free-energy landscape can be perturbed experimentally as a function of pressure. Specifically, the amplitudes of individual kinetic phases that were observed in stopped-flow studies of the hydride transfer reaction are sensitive to pressure; this indicates that pressure drives an altered distribution across the energy landscape. We show by absorbance spectroscopy that the loss of charge-transfer character of the enzyme-coenzyme complex is attributed to the altered population of MRCs on the landscape. The existence of a conformationally rich landscape in the N189A mutant is supported by molecular dynamics simulations at low and high pressure. The work provides firm experimental and computational support for the existence of parallel pathways arising from multiple conformational states of the enzyme-coenzyme complex. Hydrostatic pressure is a powerful and general probe of multidimensional energy landscapes that can be used to analyse experimentally parallel pathways for enzyme-catalysed reactions. We suggest that this is especially the case following directed mutation of a protein, which can lead to increased population of reactant states that are essentially inaccessible in the free-energy landscape of wild-type enzyme.
- Published
- 2009
25. Computational Simulations of Tunnelling Reactions in Enzymes
- Author
-
Jiayun Pang, Nigel S. Scrutton, and Michael J. Sutcliffe
- Subjects
Chemistry ,Chemical physics ,Computational chemistry ,Hydrogen transfer ,Quantum tunnelling - Abstract
The simulation of hydrogen transfer in enzymes provides a uniquely detailed atomistic insight into the reaction. Despite recent advances, such simulations remain challenging due to the importance of both electronic and nuclear quantum effects as well as the effects of protein motion. The incorporati...
- Published
- 2009
26. Secondary kinetic isotope effects as probes of environmentally-coupled enzymatic hydrogen tunneling reactions
- Author
-
Rhiannon M. Evans, Nigel S. Scrutton, Rudolf Konrad Allemann, Xi Wang, Michael J. Sutcliffe, Jiayun Pang, Sam Hay, and Phillip J. Monaghan
- Subjects
Hydrogen ,Inorganic chemistry ,chemistry.chemical_element ,Photochemistry ,Catalysis ,Enzyme catalysis ,Geobacillus stearothermophilus ,Isotopes ,Kinetic isotope effect ,Dihydrofolate reductase ,Thermotoga maritima ,Physical and Theoretical Chemistry ,Peptide Synthases ,Morphinone reductase ,biology ,Hydride ,Alcohol Dehydrogenase ,Active site ,NAD ,Atomic and Molecular Physics, and Optics ,Kinetics ,chemistry ,Models, Chemical ,Molecular Probes ,biology.protein ,Quantum Theory ,Oxidation-Reduction ,NADP - Abstract
The secondary kinetic isotope effect for hydride transfer from NADPH to dihydrofolate catalyzed by dihydrofolate reductase (see traces) is neither temperature dependent nor exalted. In environmentally coupled models of H-tunneling, the secondary isotope effects do not report on promoting motions, but reflect the active site geometry attained immediately prior to H transfer (i.e. the ‘tunnelling ready configuration′).
- Published
- 2008
27. Mutagenesis of morphinone reductase induces multiple reactive configurations and identifies potential ambiguity in kinetic analysis of enzyme tunneling mechanisms
- Author
-
Jiayun Pang, Sam Hay, Michael J. Sutcliffe, Nigel S. Scrutton, David Leys, Christopher R. Pudney, and Claire Costello
- Subjects
Models, Molecular ,Morphinone reductase ,Chemistry ,Hydride ,Hydrogen bond ,Stereochemistry ,Hydrogen Bonding ,General Chemistry ,Crystal structure ,Ring (chemistry) ,Crystallography, X-Ray ,NAD ,Biochemistry ,Catalysis ,Molecular dynamics ,Crystallography ,Kinetics ,Colloid and Surface Chemistry ,Bacterial Proteins ,Models, Chemical ,Kinetic isotope effect ,Moiety ,Oxidoreductases - Abstract
We have identified multiple reactive configurations (MRCs) of an enzyme-coenzyme complex that have measurably different kinetic properties. In the complex formed between morphinone reductase (MR) and the NADH analogue 1,4,5,6-tetrahydro-NADH (NADH4) the nicotinamide moiety is restrained close to the FMN isoalloxazine ring by hydrogen bonds from Asn-189 and His-186 as determined from the X-ray crystal structure. Molecular dynamic simulations indicate that removal of one of these hydrogen bonds in the N189A MR mutant allows the nicotinamide moiety to occupy a region of configurational space not accessible in wild-type enzyme. Using stopped-flow spectroscopy, we show that reduction of the FMN cofactor by NADH in N189A MR is multiphasic, identifying at least four different reactive configurations of the MR-NADH complex. This contrasts with wild-type MR in which hydride transfer occurs by environmentally coupled tunneling in a single kinetic phase [Pudney et al. J. Am. Chem. Soc. 2006, 128, 14053-14058]. Values for primary and alpha-secondary kinetic isotope effects, and their temperature dependence, for three of the kinetic phases in the N189A MR are consistent with hydride transfer by tunneling. Our analysis enables derivation of mechanistic information concerning different reactive configurations of the same enzyme-coenzyme complex using ensemble stopped-flow methods. Implications for the interpretation from kinetic data of tunneling mechanisms in enzymes are discussed.
- Published
- 2007
28. Molecular dynamics simulation of thermal unfolding of Thermatoga maritima DHFR
- Author
-
Jiayun Pang and Rudolf Konrad Allemann
- Subjects
Protein Denaturation ,Protein Folding ,Time Factors ,Stereochemistry ,Protein Conformation ,Protein subunit ,Dimer ,General Physics and Astronomy ,Protein Structure, Secondary ,chemistry.chemical_compound ,Protein structure ,Dihydrofolate reductase ,Escherichia coli ,Computer Simulation ,Thermotoga maritima ,Physical and Theoretical Chemistry ,Protein secondary structure ,biology ,Temperature ,Hyperthermophile ,Folding (chemistry) ,Tetrahydrofolate Dehydrogenase ,chemistry ,Models, Chemical ,biology.protein ,Protein folding ,Dimerization - Abstract
Molecular dynamics simulations of the temperature-induced unfolding reaction of native dimeric dihydrofolate reductase from the hyperthermophile Thermatoga maritima (TmDHFR) and the experimentally inaccessible TmDHFR monomer were carried out at 400 K, 450 K and 500 K. The results revealed that the unfolding of TmDHFR subunits followed a similar path to that of the monomeric DHFR from the mesophile E. coli (EcDHFR). An initial collapse of the adenosine-binding domain (ABD) was followed by the loss of the N-terminal and loop domains (NDLD). Interestingly, the elements of the secondary structure of the isolated TmDHFR monomer were maintained for significantly longer periods of time for the hyperthermophilic enzyme, suggesting that subunit stability contributes to the enhanced resistance of TmDHFR to temperature-induced unfolding. The interactions between the subunits of the TmDHFR dimer led to a stabilisation of the NDLD. The hydrogen bonds between residues 140-143 in betaG of one subunit and residues 125-127 in betaF of the other subunit were retained for significant parts of the simulations at all temperatures. These intermolecular hydrogen bonds were lost after the unfolding of the individual subunits. The high stability of the dimer mediated by strong intersubunit contacts together with an intrinsically enhanced stability of the subunits compared to EcDHFR provides a molecular rational for the higher stability of the thermophilic enzyme. The computed unfolding pathways suggest that the partly folded dimer may be a genuine folding intermediate.
- Published
- 2007
29. Protein motions during catalysis by dihydrofolate reductases
- Author
-
Paul J. Shrimpton, Rudolf Konrad Allemann, Richard S. Swanwick, Jiayun Pang, Giovanni Maglia, Robert J. Rodriguez, Rhiannon M. Evans, and Lai-Hock Tey
- Subjects
Models, Molecular ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Catalysis ,Enzyme catalysis ,Evolution, Molecular ,Bacterial Proteins ,Species Specificity ,Dihydrofolate reductase ,medicine ,Escherichia coli ,chemistry.chemical_classification ,biology ,Protein dynamics ,Temperature ,Active site ,biology.organism_classification ,Kinetics ,Tetrahydrofolate Dehydrogenase ,Enzyme ,chemistry ,Biochemistry ,Thermotoga maritima ,Mutation ,biology.protein ,General Agricultural and Biological Sciences ,Hydrogen ,Research Article - Abstract
Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima . We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.
- Published
- 2006
30. Hydride transfer reaction catalyzed by hyperthermophilic dihydrofolate reductase is dominated by quantum mechanical tunneling and is promoted by both inter- and intramonomeric correlated motions
- Author
-
Donald G. Truhlar, Jiali Gao, Jingzhi Pu, Jiayun Pang, and Rudolf Konrad Allemann
- Subjects
Models, Molecular ,Stereochemistry ,Dimer ,Biochemistry ,Catalysis ,Enzyme catalysis ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Reaction rate constant ,Kinetic isotope effect ,Escherichia coli ,Thermotoga maritima ,Binding Sites ,Hydride ,Hydrogen Bonding ,General Chemistry ,Deuterium ,Crystallography ,Tetrahydrofolate Dehydrogenase ,Monomer ,chemistry ,Energy Transfer ,Quantum Theory ,Thermodynamics ,Protein quaternary structure ,Algorithms - Abstract
Simulations of hydride and deuteride transfer catalyzed by dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) are presented. TmDHFR was modeled with its active homodimeric quaternary structure, where each monomer has three subdomains. The potential energy function was a combined quantum mechanical and molecular mechanical potential (69 atoms were treated quantum mechanically, and 35 287, by molecular mechanics). The calculations of the rate constants by ensemble-averaged variational transition state theory with multidimensional tunneling predicted that hydride and deuteride transfer at 278 K proceeded with 81 and 80% by tunneling. These percentages decreased to 50 and 49% at 338 K. The kinetic isotope effect was dominated by contributions of bound vibrations and decreased from 3.0 to 2.2 over the temperature range. The calculated rates for hydride and deuteride transfer catalyzed by the hypothetical monomer were smaller by approximately 2 orders of magnitude. At 298 K tunneling contributed 73 and 66% to hydride and deuteride transfer in the monomer. The decreased catalytic efficiency of the monomer was therefore not the result of a decrease of the tunneling contribution but an increase in the quasi-classical activation free energy. The catalytic effect was associated in the dimer with correlated motions between domains as well as within and between subunits. The intrasubunit correlated motions were decreased in the monomer when compared to both native dimeric TmDHFR and monomeric E. coli enzyme. TmDHFR and its E. coli homologue involve similar patterns of correlated interactions that affect the free energy barrier of hydride transfer despite only 27% sequence identity and different quaternary structures.
- Published
- 2006
31. Evidence To Support the Hypothesis That Promoting Vibrations Enhance the Rate of an Enzyme Catalyzed H-Tunneling Reaction
- Author
-
David Leys, Jiayun Pang, Sam Hay, Christopher R. Pudney, Nigel S. Scrutton, Colin Levy, and Michael J. Sutcliffe
- Subjects
chemistry.chemical_classification ,Flavin Mononucleotide ,Stereochemistry ,Direct evidence ,Hydride ,Chemistry ,Substrate (chemistry) ,General Chemistry ,Biochemistry ,Catalysis ,Enzymes ,Vibration ,Kinetics ,Transition state theory ,Colloid and Surface Chemistry ,Oxidoreductase ,Chemical physics ,Molecular vibration ,Biocatalysis ,Quantum tunnelling - Abstract
In recent years there has been a shift away from transition state theory models for H-transfer reactions. Models that incorporate tunneling as the mechanism of H-transfer are now recognized as a better description of such reactions. Central to many models of H-tunneling is the notion that specific vibrational modes of the protein and/or substrate can increase the probability of a H-tunneling reaction, modes that are termed promoting vibrations. Thus far there has been limited evidence that promoting vibrations can increase the rate of H-transfer. In the present communication we examine the single hydride transfer from both NADPH and NADH to FMN in the reductive half-reaction of pentaerythritol tetranitrate reductase (PETNR). We find that there is a significant promoting vibration with NADPH but not with NADH and that the observed rate of hydride transfer is significantly (approximately 15x) faster with NADPH. We rule out differences in rate due to variation in driving force and the donor-acceptor distance, suggesting it is the promoting vibration with NADPH that is the origin of the increased observed rate. This study therefore provides direct evidence that promoting vibrations can lead to an increase in rate.
- Published
- 2009
32. Large-Scale Domain Conformational Change Is Coupled to the Activation of the Co-C Bond in the B12-Dependent Enzyme Ornithine 4,5-Aminomutase: A Computational Study.
- Author
-
Jiayun Pang, Xin Li, Morokuma, Keiji, Scrutton, Nigel S., and Sutcliffe, Michael J.
- Subjects
- *
CHEMICAL bonds , *ISOMERASES , *VITAMIN B12 , *ENZYMES , *COBALT , *CARBON , *CATALYSIS - Abstract
We present here an energetic and atomistic description of how d-ornithine 4,5-aminomutase (OAM), an adenosylcobalamin (AdoCbl; coenzyme B12)-dependent isomerase, employs a large-scale protein domain conformational change to orchestrate the homolytic rupture of the Co-C bond. Our results suggest that in going from the open form (catalytically inactive) to the closed form (catalytically active), the Rossmann domain of OAM effectively approaches the active site as a rigid body. It undergoes a combination of a ∼52° rotation and a ∼14 Å translation to bring AdoCbl-initially positioned ∼25 Å away—into the active-site cavity. This process is coupled to repositioning of the Ado moiety of AdoCbl from the eastern conformation to the northern conformation. Combined quantum mechanics and molecular mechanics calculations further indicate that in the open form, the protein environment does not impact significantly on the Co-C bond homolytic rupture, rendering it unusually stable, and thus catalytically inactive. Upon formation of the closed form, the Co-C bond is activated through the synergy of steric and electrostatic effects arising from tighter interactions with the surrounding enzyme. The more pronounced effect of the protein in the closed form gives rise to an elongated Co-C bond (by 0.03 Å), puckering of the ribose and increased "strain" energy on the Ado group and to a lesser extent the corrin ring. Our computational studies reveal novel strategies employed by AdoCbl-dependent enzymes in the control of radical catalysis. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
33. Atomistic insight into the origin of the temperature-dependence of kinetic isotope effects and H-tunnelling in enzyme systems is revealed through combined experimental studies and biomolecular simulation.
- Author
-
Sam Hay, Christopher Pudney, Parvinder Hothi, Linus O. Johannissen, Laura Masgrau, Jiayun Pang, David Leys, Michael J. Sutcliffe, and Nigel S. Scrutton
- Subjects
ENZYME kinetics ,COMPUTER simulation ,BIOLOGY experiments ,CRYSTALLOGRAPHY ,TEMPERATURE effect ,SOLUTION (Chemistry) ,CATALYSIS ,MOLECULAR dynamics - Abstract
The physical basis of the catalytic power of enzymes remains contentious despite sustained and intensive research efforts. Knowledge of enzyme catalysis is predominantly descriptive, gained from traditional protein crystallography and solution studies. Our goal is to understand catalysis by developing a complete and quantitative picture of catalytic processes, incorporating dynamic aspects and the role of quantum tunnelling. Embracing ideas that we have spearheaded from our work on quantum mechanical tunnelling effects linked to protein dynamics for H-transfer reactions, we review our recent progress in mapping macroscopic kinetic descriptors to an atomistic understanding of dynamics linked to biological H-tunnelling reactions. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
34. Protein motions during catalysis by dihydrofolate reductases.
- Author
-
Rudolf K. Allemann, Rhiannon M. Evans, Lai-hock Tey, Giovanni Maglia, Jiayun Pang, Robert Rodriguez, Paul J. Shrimpton, and Richard S. Swanwick
- Published
- 2006
35. Hydride Transfer Reaction Catalyzed by Hyperthermophilic Dihydrofolate Reductase Is Dominated by Quantum Mechanical Tunneling and Is Promoted by Both Inter- and Intramonomeric Correlated Motions.
- Author
-
Jiayun Pang, Jingzhi Pu, Jiali Gao, Truhlar, Donald G., and Allemann, Rudolf K.
- Subjects
- *
HYDRIDES , *QUANTUM tunneling , *QUANTUM theory , *ESCHERICHIA coli , *ENZYME kinetics , *MOLECULAR dynamics , *MONOMERS - Abstract
Simulations of hydride and deuteride transfer catalyzed by dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) are presented. TmDHFR was modeled with its active homodimeric quaternary structure, where each monomer has three subdomains. The potential energy function was a combined quantum mechanical and molecular mechanical potential (69 atoms were treated quantum mechanically, and 35 287, by molecular mechanics). The calculations of the rate constants by ensemble-averaged variational transition state theory with multidimensional tunneling predicted that hydride and deuteride transfer at 278 K proceeded with 81 and 80% by tunneling. These percentages decreased to 50 and 49% at 338 K. The kinetic isotope effect was dominated by contributions of bound vibrations and decreased from 3.0 to 2.2 over the temperature range. The calculated rates for hydride and deuteride transfer catalyzed by the hypothetical monomer were smaller by approximately 2 orders of magnitude. At 298 K tunneling contributed 73 and 66% to hydride and deuteride transfer in the monomer. The decreased catalytic efficiency of the monomer was therefore not the result of a decrease of the tunneling contribution but an increase in the quasi-classical activation free energy. The catalytic effect was associated in the dimer with correlated motions between domains as well as within and between subunits. The intrasubunit correlated motions were decreased in the monomer when compared to both native dimeric TmDHFR and monomeric E. coli enzyme. TmDHFR and its E. coli homologue involve similar patterns of correlated interactions that affect the free energy barrier of hydride transfer despite only 27% sequence identity and different quaternary structures. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
36. Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines MECHANISTIC INSIGHTS FROM ISOTOPE EFFECTS AND QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIPS.
- Author
-
Heuts, Dominic P. H. M., Gummadova, Jennet O., Jiayun Pang, Rigby, Stephen E. J., and Scrutton, Nigel S.
- Subjects
- *
AMINES , *ISOTOPES , *LEUCOCYTES , *PHENETHYLAMINES , *QUINONE - Abstract
Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pKa values that defined a bell-shaped plot of turnover number kcat,app as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (kcat/Km)app on pH revealed a single pKa value (9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (kcat/Km)app over the pH range of 6 to 10 was observed with d2-benzylamine. Over the same pH range, the KIE on kcat was found to be close to unity. The unusual KIE values on (kcat/Km)app were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on kcat,app (8.01 ± 0.28 with phenylethylamine), indicating that C-H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
37. Evidence To Support the Hypothesis That Promoting Vibrations Enhance the Rate of an Enzyme Catalyzed H-Tunneling Reaction.
- Author
-
Pudney, Christopher R., Hay, Sam, Levy, Colin, Jiayun Pang, Sutcliffe, Michael J., Leys, David, and Scrutton, Nigel S.
- Subjects
- *
ORGANONITROGEN compounds , *ENZYMES , *FLAVINS , *COENZYMES , *PENTAERYTHRITOL tetranitrate , *DETONATORS - Abstract
The article examines the origin of rate enhancement of flavin mono-nucleotide (FMN). It states that FMN reduction can be monitored as a pseudo-first-order reaction using single-turnover approaches. It suggests that the difference in observed rate between two near identical coenzymes is related to a vibrational mode present in the pentaerythritol tetranitrate reductase (PETNR) NADPH complex.
- Published
- 2009
- Full Text
- View/download PDF
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