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Enzyme Interaction (enzyme + interaction)
Selected AbstractsInsecticide resistance spectra and resistance mechanisms in populations of Japanese encephalitis vector mosquitoes, Culex tritaeniorhynchus and Cx. gelidus, in Sri LankaMEDICAL AND VETERINARY ENTOMOLOGY, Issue 4 2000S. H. P. P. Karunaratne Summary Culex tritaeniorhynchus Giles and Cx. gelidus Theobald (Diptera: Culicidae), both vectors of Japanese encephalitis, were collected in 1984 and 1998 from two disease endemic localities in Sri Lanka: Anaradhapura and Kandy. Using wild-caught adult mosquitoes from light traps, log dosage-probit mortality curves for insecticide bioassays were obtained for three insecticides: malathion (organophosphate), propoxur (carbamate) and permethrin (pyrethroid). LD50 values showed that, in 1998, Cx. tritaeniorhynchus was ,100-fold more resistant to malathion and 10-fold more resistant to propoxur than was Cx. gelidus. This difference was attributed to Cx. tritaeniorhynchus breeding mostly in irrigated rice paddy fields, where it would have been exposed to pesticide selection pressure, whereas Cx. gelidus breeds in other types of aquatic habitats less prone to pesticide applications. Resistance in Cx. tritaeniorhynchus increased between 1984 and 1998, whereas Cx. gelidus remained predominantly susceptible. Propoxur inhibition of acetylcholinesterase (AChE) activity (the target site of organophosphates and carbamates) indicated that in 1998, frequencies of insensitive AChE-based resistance were 9% in Cx. gelidus and 2,23% in Cx. tritaeniorhynchus, whereas in 1984 this resistance mechanism was detected only in 2% of the latter species from Anaradhapura. The AChE inhibition coefficient (ki) with propoxur was 1.86 ± 0.24 × 105 m,1 min,1 for Cx. tritaeniorhynchus from Anaradhapura in 1998. Both species were tested for activity levels of detoxifying glutathione S-trans- ferases (GSTs) and malathion-specific as well as general carboxylesterases. High activities of GSTs and carboxylesterases were detected in Cx. tritaeniorhynchus but not Cx. gelidus. Malathion-specific carboxylesterase was absent from both species. Native polyacrylamide gel electrophoresis resolved two elevated general carboxylesterases, CtrEst,1 and CtrEst,1, from Cx. tritaeniorhynchus and none from Cx. gelidus. CtrEst,1 was the most intensely staining band. Gel inhibition experiments showed that both elevated esterases were inhibited by organophosphates and carbamates but not by pyrethroids. The major elevated esterase CtrEst,1 was partially purified (15-fold) by sequential Q-Sepharose and phenyl Sepharose column chromatography. The bimolecular rate constant (ka) and the deacylation rate constant (k3) for the malaoxon/ enzyme interaction were 9.9 ± 1.1 × 103 m,1 min,1 and 3.5 ± 0.05 × 10,4m,1 min,1, respectively, demonstrating that the role of this enzyme in organophosphorus insecticide resistance is sequestration. [source] Scalaradial, a Dialdehyde-Containing Marine Metabolite That Causes an Unexpected Noncovalent PLA2 InactivationCHEMBIOCHEM, Issue 13 2007Maria Chiara Monti Dr. Abstract Several marine terpenoids that contain at least one reactive aldehyde group, such as manoalide and its congeners, possess interesting anti-inflammatory activities that are mediated by the covalent inactivation of secretory phospholipase A2 (sPLA2). Scalaradial, a 1,4-dialdehyde marine terpenoid that was isolated from the sponge Cacospongia mollior, is endowed with a relevant anti-inflammatory profile, both in vitro and in vivo, through selective sPLA2 inhibition. Due to its peculiar dialdehyde structural feature, it has been proposed that scalaradial exerts its enzymatic inactivation by means of an irreversible covalent modification of its target. In the context of our on-going research on anti-PLA2 natural products and their interaction at a molecular level, we studied scalaradial in an attempt to shed more light on the molecular mechanism of its PLA2 inhibition. A detailed analysis of the reaction profile between scalaradial and bee venom PLA2, a model sPLA2 that shares a high structural homology with the human synovial enzyme, was performed by a combination of spectroscopic techniques, chemical reactions (selective modifications, biomimetic reactions), and classical protein chemistry (such as proteolytic digestion, HPLC and mass spectrometry), along with molecular modeling studies. Unexpectedly, our data clearly indicated the noncovalent forces to be the leading event in the PLA2 inactivation process; thus, the covalent modification of the enzyme emerges as only a minor side event in the ligand,enzyme interaction. The overall picture might be useful in the design of SLD analogues as new potential anti-inflammatory compounds that target sPLA2 enzymes. [source] Thermodynamic analysis of binding of p -substituted benzamidines to trypsinFEBS JOURNAL, Issue 6 2001Reinskje Talhout Understanding the structural basis of inhibitor,enzyme interactions, important for the design of new drugs, requires a complete thermodynamic characterization of the binding process as well as a description of the structure of the complex. In this paper, the binding of p -substituted benzamidinium derivatives to the structurally well-characterized serine proteinase bovine pancreatic trypsin has been studied using isothermal titration calorimetry. These experiments have permitted a complete characterization of the temperature dependence of the inhibitor-binding thermodynamics. At 25 °C, both the enthalpy and entropy of binding are favourable for all studied derivatives, but this is only true for a relatively narrow temperature range. As binding is characterized by a negative change in heat capacity, the process is characterized by enthalpy,entropy compensation, resulting in a change of the net thermodynamic driving force for association from entropic to enthalpic with increasing temperature. These phenomena are not unusual when hydrophobic forces play an important role. The trend in the relative binding potencies can, to a significant extent, be attributed to the electron-donating/withdrawing character of the substituent at the para position, as shown by the Hammett plot for the different inhibitors; the more polar the p -substituted benzamidine, the less potent it will be as a trypsin inhibitor. This behaviour might result from a bulk solvation effect, meaning that the more polar, lower potency inhibitors will be more stabilized in water than the less polar, higher potency inhibitors. [source] Ab initio vibrational calculations on ara-T molecule: Application to analysis of IR and Raman spectraBIOPOLYMERS, Issue 4 2001Belén Hernández Abstract The FTIR and FT-Raman spectra are reported for the arabinonucleoside ara-T (1-,- D -arabinofuranosylthymine), which shows antiviral activity. The accurate knowledge of the vibrational modes is a prerequisite for the elucidation of drug,nucleotide and drug,enzyme interactions. The FTIR and FT-Raman spectra of ara-T were recorded from 4000 to 30 cm,1. A tetradeuterated derivative (deuteration at N3, and hydroxyl groups O,2, O,3, and O,5) was synthesized and the observed isotopic shifts in its spectra were used for the vibrational analysis of ara-T. The theoretical frequencies and the potential energy distribution (PED) of the vibrational modes of ara-T were calculated using the ab initio Hartree,Fock/3-21G method. An assignment of the vibrational spectra of ara-T is proposed considering the scaled PED and the observed band shifts under deuteration. The scaled ab initio frequencies were in reasonable agreement with the experimental data. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 193,207, 2001 [source] Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the proteinPROTEIN SCIENCE, Issue 6 2000Richard GrießLer Abstract Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5,-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (,50°C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate,16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the ,-helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5,-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to, 50°C, and (2) stabilizes the covalent protein cofactor linkage up to , 70°C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50°C of , 25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a , 20°C higher value on the temperature scale. [source] | ||||||||||