933 resultados para Active-site binding specificity
Resumo:
The coronavirus main protease, Mpro, is considered a major target for drugs suitable to combat coronavirus infections including the severe acute respiratory syndrome (SARS). In this study, comprehensive HPLC- and FRET-substrate-based screenings of various electrophilic compounds were performed to identify potential Mpro inhibitors. The data revealed that the coronaviral main protease is inhibited by aziridine- and oxirane-2-carboxylates. Among the trans-configured aziridine-2,3-dicarboxylates the Gly-Gly-containing peptide 2c was found to be the most potent inhibitor.
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KF, LiF and CsF/A(2)O(3) catalysts with different loadings from 1 to 20 wt% were prepared using aqueous solutions of the alkaline fluoride compounds by wet impregnation of basic mesoporous MSU-type alumina. The catalysts were activated under At at 400 degrees C for 2 h and monitored by in situ XRD measurements. The catalysts were also characterized using several techniques: N-2 adsorption/desorption isotherms at -196 degrees C, FTIR, DR-UV-vis, CO2-TPD, XRD, Al-27 CP/MAS NMR. These characterizations led to the conclusion that the deposition of alkaline fluorides on the alumina surface generates fluoroaluminates and aluminate species. The process is definitivated at 400 degrees C. The fluorine in these structures is less basic than in the parent fluorides, but the oxygen becomes more basic. The catalysts were tested for the transesterification of fatty esters under different experimental conditions using conventional heating, microwave and Ultrasound irradiation. Recycling experiments showed that these catalysts are stable for a limited number of cycles. (C) 2009 Elsevier Inc. All rights reserved.
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A molecular model for the P450 enzyme cytochrome P450 C17 (CYP17) is presented based on sequence alignments of multiple template structures and homology modeling. This enzyme plays a central role in the biosynthesis of testosterone and is emerging as a major target in prostate cancer, with the recently developed inhibitor abiraterone currently in advanced clinical trials. The model is described in detail, together with its validation, by providing structural explanations to available site-directed mutagenesis data. The CYP17 molecule in this model is in the form of a triangular prism, with an edge of similar to 55 angstrom and a thickness of similar to 37 angstrom. It is predominantly helical, comprising 13 alpha helices interspersed by six 3(10) helices and 11 beta-sheets. Multinanosecond molecular dynamics simulations in explicit solvent have been carried out, and principal components analysis has been used to reveal the details of dynamics around the active site. Coarse-grained methods have also been used to verify low-frequency motions, which have been correlated with active-site gating. The work also describes the results of docking synthetic inhibitors, including the drug abiraterone and the natural substrate pregnenolone, in the CYP17 active site together with molecular dynamics simulations on the complexes. (C) 2010 Elsevier Ltd. All rights reserved.
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Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C-1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the gamma-phosphorus of ATP.
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A novel [Ni'S-4'Fe-2(CO)(6)] cluster (1: 'S-4'=(CH3C6H3S2)(2)(CH2)(3)) has been synthesised, structurally characterised and has been shown to undergo a chemically reversible reduction process at -1.31 V versus Fc(+)/Fc to generate the EPR-active monoanion 1(-). Multifrequency Q-, X- and S-band EPR spectra of Ni-61-enriched 1(-) show a well-resolved quartet hyperfine splitting in the low-field region due to the interaction with a single Ni-61 (I = 3/2) nucleus. Simulations of the EPR spectra require the introduction of a single angle of non-coincidence between g, and A(1), and g(3) and A(3) to reproduce all of the features in the S- and X-band spectra. This behaviour provides a rare example of the detection and measurement of non-coincidence effects from frozen-solution EPR spectra without the need for single-crystal measurements, and in which the S-band experiment is sensitive to the non-coincidence. An analysis of the EPR spectra of 1(-) reveals a 24% Ni contribution to the SOMO in 1(-), supporting a delocalisation of the spin-density across the NiFe2 cluster. This observation is supported by IR spectroscopic results which show that the CO stretching frequencies, v(CO), shift to lower frequency by about 70 cm(-1) when 1 is reduced to 1(-). Density functional calculations provide a framework for the interpretation of the spectroscopic properties of 1(-) and suggest that the SOMO is delocalised over the whole cluster, but with little S-centre participation. This electronic structure contrasts with that of the Ni-A, -B, -C and -L forms of [NiFe] hydrogenase in which there is considerable S participation in the SOMO.
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Hydrogenases are enzymes that catalyse the reversible two-electron oxidation of H-2. The [NiFe] hydrogenases have been characterised spectroscopically and by single crystal X-ray diffraction, and show an active site incorporating a heterobinuclear [NiFe] centre bridged by two cysteine S-donors. Low molecular weight synthetic complex models, which structurally mimic the dithiolate-bridged [NiFe] centre, serve as important probes of structure and chemistry at the active site and are the subject of this review. (C) 2001 Elsevier Science B.V. All rights reserved.
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We designed a straightforward biotinylated probe using the N-terminal substrate-like region of the inhibitory site of human cystatin C as a scaffold, linked to the thiol-specific reagent diazomethylketone group as a covalent warhead (i.e. Biot-(PEG)2-Ahx-LeuValGly-DMK). The irreversible activity-based probe bound readily to cysteine cathepsins B, L, S and K. Moreover affinity labeling is sensitive since active cathepsins were detected in the nM range using an ExtrAvidin®-peroxidase conjugate for disclosure. Biot-(PEG)2-Ahx-LeuValGly-DMK allowed a slightly more pronounced labeling for cathepsin S with a compelling second-order rate constant for association (kass = 2,320,000 M−1 s−1). Labeling of the active site is dose-dependent as observed using 6-cyclohexylamine-4-piperazinyl-1,3,5-triazine-2-carbonitrile, as competitive inhibitor of cathepsins. Finally we showed that Biot-(PEG)2-Ahx-LeuValGly-DMK may be a simple and convenient tool to label secreted and intracellular active cathepsins using a myelomonocytic cell line (THP-1 cells) as model.
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Abstract The dehydrogenation of cyclohexanol to cyclohexanone is very important in the manufacture of nylon. Copper-based catalysts are the most popular catalysts for this reaction, and on these catalysts the reaction mechanism and active site are in debate. In order to elucidate the mechanism and active site of the cyclohexanol dehydrogenation on copper-based catalysts, density functional theory with dispersion corrections were performed on up to six facets of copper in two different oxidation states: monovalent copper and metallic copper. By calculating the surface energies of these facets, Cu(111) and Cu2O(111) were found to be the most stable facets for metallic copper and for monovalent copper, respectively. On these two facets, all the possible elementary steps in the dehydrogenation pathway of cyclohexanol were calculated, including the adsorption, dehydrogenation, hydrogen coupling and desorption. Two different reaction pathways for dehydrogenation were considered on both surfaces. It was revealed that the dehydrogenation mechanisms are different on these two surfaces: on Cu(111) the hydrogen belonging to the hydroxyl is removed first, then the hydrogen belonging to the carbon is subtracted, while on Cu2O(111) the hydrogen belonging to the carbon is removed followed by the subtraction of the hydrogen in the hydroxyl group. Furthermore, by comparing the energy profiles of these two surfaces, Cu2O(111) was found to be more active for cyclohexanol dehydrogenation than Cu(111). In addition, we found that the coordinatively unsaturated copper sites on Cu2O(111) are the reaction sites for all the steps. Therefore, the coordinatively unsaturated copper site on Cu2O(111) is likely to be the active site for cyclohexanol dehydrogenation on the copper-based catalysts.
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Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.
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PUF proteins regulate both stability and translation through sequence-specific binding to the 3` UTR of target mRNA transcripts. Binding is mediated by a conserved PUF domain, which contains eight repeats of approximately 36 amino acids each. Found in all eukaryotes, they have been related to several developmental processes. Analysis of the 25 Arabidopsis Pumilio (APUM) proteins presenting PUF repeats reveals that 12 (APUM-1 to APUM-12) have a PUF domain with 50-75% similarity to the Drosophila PUF domain. Through three-hybrid assays, we show that APUM-1 to APUM-6 can bind specifically to the Nanos response element sequence recognized by Drosophila Pumilio. Using an Arabidopsis RNA library in a three-hybrid screening, we were able to identify an APUM-binding consensus sequence. Computational analysis allowed us to identify the APUM-binding element within the 3` UTR in many Arabidopsis transcripts, even in important mRNAs related to shoot stem cell maintenance. We demonstrate that APUM-1 to APUM-6 are able to bind specifically to APUM-binding elements in the 3` UTR of WUSCHEL, CLAVATA-1, PINHEAD/ZWILLE and FASCIATA-2 transcripts. The results obtained in the present study indicate that the APUM proteins may act as regulators in Arabidopsis through an evolutionarily conserved mechanism, which may open up a new approach for investigating mRNA regulation in plants.
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This study answers several pending questions about alumina-catalyzed epoxidation with aqueous 70 wt% H2O2. To evaluate the effect of the water-to-aluminum tri-sec-butoxide molar ratio, this was systematically changed from 1 to 24. The xerogels were calcined at 450 degrees C and gave different gamma-Al2O3's with distinct textural and acidic properties. A combination of Al-27 MAS NMR and TPD-NH3 results of calcined aluminas allowed us to assign the type la. Al-OH sites as the catalytic sites for epoxidation. The type Ib Al-OH sites have no function in catalytic epoxidation, because ethyl acetate poisons these sites. The strong acid sites of types IIa, IIb, and III Al-OH groups are responsible for the undesired H2O2 decomposition and decreased oxidant selectivity. (c) 2006 Elsevier B.V. All rights reserved.