Home About us Contact
|
Home About us Contact | ||
|
|
||
Active Site Serine (active + site_serine)
Selected AbstractsDichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdoseJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2010Bin Li Abstract The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60,84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase. Copyright © 2010 John Wiley & Sons, Ltd. [source] Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2004Martin Zehl Abstract A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H]2+ ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme. Copyright © 2004 John Wiley & Sons, Ltd. [source] Factor Xa is highly protected from antithrombin,fondaparinux and antithrombin,enoxaparin when incorporated into the prothrombinase complexJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2003N. Brufatto Summary., Antithrombin and its cofactor, heparin, target both the product of prothrombin activation by prothrombinase, thrombin, as well as the enzyme responsible for the reaction, factor (F)Xa. These studies were carried out to quantify the effects of each of the prothrombinase components on the half-life of FXa in the presence of antithrombin and the low-molecular-weight heparins (enoxaparin, Aventis, Laval, Quebec, Canada) or the heparin pentasaccharide (fondaparinux, Organon Sanofi-Synthelabo, Cypress, TX, USA). Experiments were carried out using a recombinant form of prothrombin in which the active site serine has been mutated to cysteine and subsequently labeled with fluorescein. This mutant allowed calculation of the second order rate constant for inhibition of FXa by antithrombin in such a way that competition for antithrombin by thrombin is eliminated and competition for FXa by prothrombin is accounted for. Intrinsic rate constants for the inhibition of FXa by antithrombin,enoxaparin and antithrombin,fondaparinux, in the presence of the various prothrombinase components, were calculated. Addition of phospholipid had no significant effect on the second order rate constant for inhibition of FXa by antithrombin, while addition of FVa appeared to be mildly protective. Further addition of prothrombin however, caused profound protection of FXa, increasing its half-life from 1.1 to 353 s in the case of fondaparinux, and from 0.4 to 42 s in the case of enoxaparin. Similar results were reported for unfractionated heparin previously [1]. Therefore, in the presence of unfractionated heparin, fondaparinux, or enoxaparin, prothrombinase is profoundly protected from antithrombin. [source] QSAR for Inhibition of Pseudomonas Species Lipase by 1-Acyloxy-3- N-n -octylcarbamyl-benzenesMOLECULAR INFORMATICS, Issue 3 2009Shyh-Ying Chiou Abstract 1-Acyloxy-3- N-n -octylcarbamyl-benzenes (1,9) are synthesized to characterize the Quantitative Structure,Activity Relationship (QSAR) for the Third Acyl Group Binding Site (TACS) of Pseudomonas species lipase. Inhibitors 1,9 are characterized as pseudo or alternate substrate inhibitors of the enzyme. The inhibition constant (Ki) and carbamylation constant (k2) for the enzyme inhibitions by inhibitors 1,9 are determined. The carbamate carbons of the n -octylcarbamyl moieties of inhibitors 1,9 are nucleophilically attacked by the active site serine of the enzyme and the n -octylcarbamyl groups of inhibitors 1,9 are bound to the Acyl Group Binding Site (ACS) of the enzyme. Both pKi and log,k2 values are linearly corrected with the Hansch hydrophobicity , values of the substituents of the acyl moieties of inhibitors 1,7. The slopes for these corrections are 0.13 and 0.02, respectively. This result suggests that the enzyme inhibitions by inhibitors 1,7 have a common mechanism. Thus, all acyl moieties of inhibitors 1,7 should bind to the TACS of the enzyme since the acyl and carbamyl moieties of inhibitors 1,7 are meta to each other. This result also indicates that the major interaction between the acyl moiety of inhibitors 1,7 and the TACS of the enzyme is primarily the hydrophobic interaction. The more hydrophobic characters of inhibitors 1,7 are, the more tightly these inhibitors bind to the enzyme. In contrast, 1-triphenylacetoxy-3- N-n -octylcarbamyl-benzene (8) and 1-trimethylacetoxy-3- N-n -octylcarbamyl-benzene (9) do not bind to the TACS of the enzyme due to the fact that the inhibitions by both inhibitors are not linearly correlated with ,. It is possible that these two inhibitors are too bulky to fit into the TACS of the enzyme. [source] Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activityJOURNAL OF MOLECULAR RECOGNITION, Issue 5 2008Rui Huo Abstract In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t -butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH,+,VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni2+ -immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0,3.0,mg of proteins from 1,L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288,U/µmol. Copyright © 2008 John Wiley & Sons, Ltd. [source] | ||||||||||