Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.
Two-dimensional (2D) 1 H-31 P NMR techniques are applied to samples containing trace amounts of organophosphorus (OP) compounds related to the Chemical Weapons Convention. Because these techniques allow the recording of 31 P-filtered spectra with the sensitivity of proton NMR, the problem of overlapping in 1 H NMR spectra is nearly canceled, while simultaneous 31 P and 1 H NMR information is obtained in a fast way. How these experiments are carried out is described. The advantages of 2D techniques are compared with respect to their 1D counterparts, and new developments are proposed for rapid and reliable assignment without the need for reference data of the spiked OP chemicals.
The three-dimensional structure of bovine angiogenin has been determined using two- and three-dimensional proton NMR spectroscopy. The solution structure is very close to that recently determined by X-ray diffraction analysis. This structure appears well defined, even if five loops and one helix exhibit greater flexibility. Analysis of the active site geometry confirms the position of the Glu-118 residue which obstructs the pyrimidine binding site. There is no experimental evidence of an unobstructed conformation of angiogenin in solution. In addition, it appears that the Glu-118 and Ser-119 residues and the cell receptor binding loop may play an important role in the differences of C-terminal fragment organization and ribonucleolytic activity observed between angiogenins and ribonuclease A.
Novel 2,3-heterodisubstituted β-cyclodextrin derivatives were designed as artificial enzymes to degrade chemical warfare agents. One of them reduced the acetylcholinesterase inhibitory potential by soman faster than its monosubstituted analog.
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