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171.
Activation of the binuclear metal center through formation of phosphotriesterase-inhibitor complexes
Phosphotriesterase (PTE) from Pseudomonas diminuta is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphate nerve agents at rates approaching the diffusion-controlled limit. The proposed catalytic mechanism postulates the interaction of the substrate with the metal center and subsequent nucleophilic attack by the bridging hydroxide. X-band EPR spectroscopy was utilized to monitor the active site of Mn/Mn-substituted PTE upon addition of two inhibitors, diisopropyl methyl phosphonate and triethyl phosphate, and the product of hydrolysis, diethyl phosphate. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge were evaluated by measuring changes in the features of the EPR spectra. The EPR spectra support the proposal that the binding of substrate analogues to the binuclear metal center diminishes the population of hydroxide-bridged species. These results, in conjunction with previously published kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the beta-metal weakens the coordination of the hydroxide bridge to the beta-metal. The weakened coordination to the beta-metal ion increases the nucleophilic character of the hydroxide and is coupled to the increase in the electrophilic character of the substrate. 相似文献
172.
Job FM van Boven Eline Tommelein Koen Boussery Els Mehuys Stefan Vegter Guy GO Brusselle Maureen PMH Rutten-van M?lken Maarten J Postma 《Respiratory research》2014,15(1):66
Background
The PHARMACOP-intervention significantly improved medication adherence and inhalation technique for patients with COPD compared with usual care. This study aimed to evaluate its cost-effectiveness.Methods
An economic analysis was performed from the Belgian healthcare payer’s perspective. A Markov model was constructed in which a representative group of patients with COPD (mean age of 70 years, 66% male, 43% current smokers and mean Forced Expiratory Volume in 1 second of % predicted of 50), was followed for either receiving the 3-month PHARMACOP-intervention or usual care. Three types of costs were calculated: intervention costs, medication costs and exacerbation costs. Outcome measures included the number of hospital-treated exacerbations, cost per prevented hospital-treated exacerbation and cost per Quality Adjusted Life-Year. Follow-up was 1 year in the basecase analysis. Sensitivity and scenario analyses (including long-term follow-up) were performed to assess uncertainty.Results
In the basecase analysis, the average overall costs per patient for the PHARMACOP-intervention and usual care were €2,221 and €2,448, respectively within the 1-year time horizon. This reflects cost savings of €227 for the PHARMACOP-intervention. The PHARMACOP-intervention resulted in the prevention of 0.07 hospital-treated exacerbations per patient (0.177 for PHARMACOP versus 0.244 for usual care). Results showed robust cost-savings in various sensitivity analyses.Conclusions
Optimization of current pharmacotherapy (e.g. close monitoring of inhalation technique and medication adherence) has been shown to be cost-saving and should be considered before adding new therapies. 相似文献173.
Rachel IM van Haaften Blanche Schroen Ben JA Janssen Arie van Erk Jacques JM Debets Hubert JM Smeets Jos FM Smits Arthur van den Wijngaard Yigal M Pinto Chris TA Evelo 《BMC bioinformatics》2006,7(1):200-15
Background
Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. 相似文献174.
175.
Distances between structural metal ion, substrates, and allosteric modifier of fructose bisphosphatase 总被引:1,自引:0,他引:1
The binding of two paramagnetic probes within a subunit of fructose bisphosphatase, viz., Mn2+ at a structural site and a nitroxide spin-label at a sulfhydryl site, has permitted the measurement of NMR and electron paramagnetic resonance (EPR) relaxation rates to map the active and allosteric site topography. Distances from these loci to the phosphoryl of fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi) and four nuclei of adenosine 5'-phosphate (AMP) (the phosphorus nucleus, H-8, H-2, and H-1') were obtained. These measurements located the Mn2+ approximately equidistant from the two phosphoryl moieties of the product ligands Fru-6-P and Pi and in close proximity to the AMP. The adenosine moiety of the latter is oriented anti. Analysis of EPR data revealed that the nitroxide group is approximately 16 A from the structural Mn2+ site. Calculation of the residence times for the hydrolysis reaction products suggests that their dissociation should not be rate limiting in the overall reaction cycle. 相似文献
176.
Andrew N. Bigley Frank M. Raushel 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):443-453
Phosphotriesters are one class of highly toxic synthetic compounds known as organophosphates. Wide spread usage of organophosphates as insecticides as well as nerve agents has lead to numerous efforts to identify enzymes capable of detoxifying them. A wide array of enzymes has been found to have phosphotriesterase activity including phosphotriesterase (PTE), methyl parathion hydrolase (MPH), organophosphorus acid anhydrolase (OPAA), diisopropylfluorophosphatase (DFP), and paraoxonase 1 (PON1). These enzymes differ widely in protein sequence and three-dimensional structure, as well as in catalytic mechanism, but they also share several common features. All of the enzymes identified as phosphotriesterases are metal-dependent hydrolases that contain a hydrophobic active site with three discrete binding pockets to accommodate the substrate ester groups. Activation of the substrate phosphorus center is achieved by a direct interaction between the phosphoryl oxygen and a divalent metal in the active site. The mechanistic details of the hydrolytic reaction differ among the various enzymes with both direct attack of a hydroxide as well as covalent catalysis being found. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases. 相似文献
177.
Dejan Maglic Karin Schlegelmilch Antonella FM Dost Riccardo Panero Raffaele A Calogero Fernando D Camargo 《The EMBO journal》2018,37(17)
The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap‐null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC. Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK‐JUN signaling, a well‐established tumor‐driving cascade. Importantly, in this context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCC pathogenesis and thereby a viable target for drug‐resistant BCC. 相似文献
178.
Positional isotope exchange 总被引:2,自引:0,他引:2
The detection of intermediates in enzyme-catalyzed reactions can be accomplished by several techniques. For those intermediates which do not have easily observed electronic spectra, use can be made of isotope exchange phenomena if the chemistry of the reaction is appropriate. Recently, the technique of positional isotope exchange (intramolecular isotopic scrambling) has been used to study several reactions which have been thought to involve high-energy intermediates in their mechanisms. A review of some of these reactions and the limitations of the method are presented in this article. 相似文献
179.
Kinetic evidence for the formation of D-alanyl phosphate in the mechanism of D-alanyl-D-alanine ligase 总被引:1,自引:0,他引:1
L S Mullins L E Zawadzke C T Walsh F M Raushel 《The Journal of biological chemistry》1990,265(16):8993-8998
The steady state kinetic mechanism, molecular isotope exchange and the positional isotope exchange (PIX) reactions of D-alanyl-D-alanine ligase from Salmonella typhimurium have been studied. The kinetic mechanism has been determined to be ordered Ter-Ter from initial velocity and product inhibition experiments. The first substrate to bind is ATP followed by the addition of 2 mol of D-alanine. Pi is released, and then D-alanyl-D-alanine and ADP dissociate from the enzyme surface. In the reverse direction D-alanyl-D-alanine exhibits complete substrate inhibition (Ki = 1.15 +/- 0.05 mM) by binding to the enzyme-ATP complex. In the presence of D-alanine, D-alanyl-D-alanine ligase catalyzed the positional exchange of the beta,gamma-bridge oxygen in [gamma-18O4]ATP to a beta-nonbridge position. Two possible alternate dead-end substrate analogs, D-2-chloropropionic acid and isobutyric acid, did not induce a positional isotope exchange in [gamma-18O4]ATP. The positional isotope exchange rate is diminished relative to the net substrate turnover as the concentration of D-alanine is increased. This is consistent with the ordered Ter-Ter mechanism as determined by the steady state kinetic experiments. The ratio of the positional isotope exchange rate relative to the net chemical turnover of substrate (Vex/Vchem) approaches a value of 1.4 as the concentration of D-alanine becomes very small. This ratio is 100 times larger than the ratio of the maximal reverse and forward chemical reaction velocities (V2/V1). This situation is only possible when the reaction mechanism proceeds in two distinct steps and the first step is much faster than the second step. The enzyme was also found to catalyze the molecular isotope exchange of radiolabeled D-alanine with D-alanyl-D-alanine in the presence of phosphate. These results are consistent with the formation of D-alanyl phosphate as a kinetically competent intermediate. 相似文献
180.
The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. Luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The time courses for the formation and decay of the various intermediates have been followed by monitoring the absorbance changes at 380 and 445 nm along with the emission of visible light using n-decanal as the alkyl aldehyde. The synthesis of the 4a-hydroperoxyflavin intermediate (FMNOOH) was monitored at 380 nm after various concentrations of luciferase, O2, and FMNH2 were mixed. The second-order rate constant for the formation of FMNOOH from the luciferase-FMNH2 complex was found to be 2.4 x 10(6) M-1 s-1. In the absence of n-decanal, this complex decays to FMN and H2O2 with a rate constant of 0.10 s-1. The enzyme-FMNH2 complex was found to isomerize prior to reaction with oxygen. The production of visible light reaches a maximum intensity within 1 s and then decays exponentially over the next 10 s. The formation of FMN from the intermediate pseudobase (FMNOH) was monitored at 445 nm. This step of the reaction mechanism was inhibited by high levels of n-decanal which indicated that a dead-end luciferase-FMNOH-decanal could form. The time courses for these optical changes have been incorporated into a comprehensive kinetic model. Estimates for 15 individual rate constants have been obtained for this model by numeric simulations of the various time courses. 相似文献